Growth hormone secretagogue receptor family

ABSTRACT

Human, swine and rat growth hormone secretagogue receptors have been isolated, cloned and sequenced. Growth hormone secretagogue receptors are new members of the G-protein family of receptors. The growth hormone secretagogue receptors may be used to screen and identify compounds which bind to the growth hormone secretagogue receptor. Such compounds may be used in the treatment of conditions which occur when there is a shortage of growth hormone, such as observed in growth hormone deficient children, elderly patients with musculoskeletal impairment and recovering from hip fracture and osteoporosis.

The instant application claims priority under 35 U.S.C. §119(e) to U.S.provisional application serial Nos. 60/008,582 and 60/018,962, filedDec. 13, 1995 and Jun. 6, 1996, respectively.

FIELD OF THE INVENTION

This invention relates to a new family of receptors, which includes thegrowth hormone secretagogue receptors (GHSRs) and growth hormonesecretagogue-related receptors (GHSRRs), nucleic acids encoding thesereceptors; and to the use of a GHSR to identify growth hormonesecretagogues and compounds that modulate GHSR function.

BACKGROUND OF THE INVENTION

Growth hormone (GH) is an anabolic hormone capable of promoting lineargrowth, weight gain and whole body nitrogen retention. Classically, GHis thought to be released primarily from the somatotroph cells of theanterior pituitary under the coordinate regulation of two hypothalamichormones, growth hormone releasing factor (GHRF or GRF) andsomatostatin. Both GHRF stimulation and somatostatin inhibition of therelease of GH occurs by the specific engagement of receptors on the cellmembrane of the somatotroph.

Recent evidence has been mounting which suggests that GH release is alsostimulated by a group of short peptides, the growth hormone releasingpeptides (GHRP; GHRP-6, GHRP-2 [hexarelin]) which are described, forexample, in U.S. Pat. No. 4,411,890, PCT Patent Pub. No. WO 89/07110,PCT Patent Pub. No. WO 89/071 11, PCT Patent Pub. No. WO 93/04081, andJ. Endocrinol Invest., 15 (Suppl 4), 45 (1992). These peptides functionby selectively binding to distinct somatotroph cell membrane receptor,the growth hormone secretagogue receptor(s) (GHSRs). A medicinalchemical approach has resulted in the design of several classes oforally-active, low molecular weight, non-peptidyl compounds which bindspecifically to this receptor and result in the pulsatile release of GH.Such compounds possessing growth hormone secretagogue activity aredisclosed, for example, in the following: U.S. Pat. Nos. 3,239,345;4,036,979; 4,411,890; 5,206,235; 5,283,241; 5,284,841; 5,310,737;5,317,017; 5,374,721; 5,430,144; 5,434,261; 5,438,136; 5,494,919;5,494,920; 5,492,916; EPO Patent Pub. No. 0,144,230; EPO Patent Pub. No.0,513,974; PCT Patent Pub. No. WO 94/07486; PCT Patent Pub. No. WO94/08583; PCT Patent Pub. No. WO 94/11012; PCT Patent Pub. No. WO94/13696; PCT Patent Pub. No. WO 94/19367; PCT Patent Pub. No. WO95/03289; PCT Patent Pub. No. WO 95/03290; PCT Patent Pub. No. WO95/09633; PCT Patent Pub. No. WO 95/11029; PCT Patent Pub. No. WO95/12598; PCT Patent Pub. No. WO 95/13069; PCT Patent Pub. No. WO95114666; PCT Patent Pub. No. WO 95/16675; PCT Patent Pub. No. WO95/16692; PCT Patent Pub. No. WO 95/17422; PCT Patent Pub. No. WO95/17423; PCT Patent Pub. No. WO 95/3431 1; PCT Patent Pub. No. WO96/02530; Science, 260, 1640-1643 (Jun. 11, 1993); Ann. Rep. Med. Chem.,28, 177-186 (1993); Bioorg. Med. Chem. Ltrs., 4(22), 2709-2714 (1994);and Proc. Natl. Acad. Sci. USA 92, 7001-7005 (July 1995).

The use of orally-active agents which stimulate the pulsatile release ofGH would be a significant advance in the treatment of growth hormonedeficiency in children and adults as well as provide substantial benefitunder circumstances where the anabolic effects of GH might be exploitedclinically (e.g. post-hip fracture rehabilitation, the frail elderly andin post-operative recovery patients).

It would also be desirable to know the molecular structure of growthhormone secretagogue receptors in order to analyze this new receptorfamily and understand its normal physiological role in concert with theactions of GHRF and somatostatin. This could lead to a betterunderstanding of the in vivo processes which occur upon ligand-receptorbinding. Further, it would be desirable to use cloned-growth hormonesecretagogue receptors as essential components of an assay system whichcan identify new growth hormone secretagogues.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to a novel family of receptors which includesgrowth hormone secretagogue receptors (GHSRs) and growth hormonesecretagogue-related receptors (GHSRRs).

A first aspect of this invention are the growth hormone secretagoguereceptors, which are free from receptor associated proteins. GHSRs maybe from any species, and in further embodiments may be isolated orpurified. One embodiment of this invention is human growth hormonesecretagogue receptor (hGHSR), free from receptor-associated proteins. Afurther aspect of this invention is hGHSR which is isolated or purified.

Another aspect of this invention is swine growth hormone secretagoguereceptor (sGHSR), free from receptor-associated proteins. A furtheraspect of this invention is sGHSR which is isolated or purified.

Another aspect of this invention is rat growth hormone secretagoguereceptor (rGHSR), free from receptor-associated proteins. A furtheraspect of this invention is sGHSR which is isolated or purified.

Another aspect of this invention are human, swine and rat GHSRs whichare encoded by substantially the same nucleic acid sequences, but whichhave undergone changes in splicing or other RNA processing-derivedmodifications or mutagenesis induced changes, so that the expressedprotein has a homologous, but different amino acid sequence from thenative forms. These variant forms may have different and/or additionalfunctions in human and animal physiology or in vitro in cell basedassays.

Another aspect of this invention are the growth hormonesecretagogue-related receptors, free from associated receptor proteins.A further embodiment are GHSRRs which are isolated or purified. Thesemay be from any species, including human, mouse, rat and swine.

Growth hormone secretagogue receptors are proteins containing variousfunctional domains, including one or more domains which anchor thereceptor in the cell membrane, and at least one ligand binding domain.As with many receptor proteins, it is possible to modify many of theamino acids, particularly those which are not found in the ligandbinding domain, and still retain at least a percentage of the biologicalactivity of the original receptor. In accordance with this invention, ithas been shown that the N-terminal portions of the GHSR are notessential for its activation by the Growth Hormone Secretagogues (GHSs).Thus this invention specifically includes modified functionallyequivalent GHSRs which have deleted, truncated, or mutated N-terminalportions. This invention also specifically includes modifiedfunctionally equivalent GHSRs which contain modified and/or deletions inother domains, which are not accompanied by a loss of functionalactivity.

Additionally, it is possible to modify other functional domains such asthose that interact with second messenger effector systems, by alteringbinding specificity and/or selectivity. Such functionally equivalentmutant receptors are also within the scope of this invention.

A further aspect of this invention are nucleic acids which encode agrowth hormone secretagogue receptor or a functional equivalent fromswine, human, rat or other species. These nucleic acids may be free fromassociated nucleic acids, or they may be isolated or purified. For mostcloning purposes, cDNA is a preferred nucleic acid, but this inventionspecifically includes other forms of DNA as well as RNAs which encode aGHSR or a functional equivalent.

Yet another aspect of this invention relates to vectors which comprisenucleic acids encoding a GHSR or a functional equivalent. These vectorsmay be comprised of DNA or RNA; for most cloning purposes DNA vectorsare preferred. Typical vectors include plasmids, modified viruses,bacteriophage and cosmids, yeast artificial chromosomes and other formsof episomal or integrated DNA that can encode a GHSR. It is well withinthe skill of the ordinary artisan to determine an appropriate vector fora particular gene transfer or other use.

A further aspect of this invention are host cells which are transformedwith a gene which encodes a growth hormone secretagogue receptor or afunctional equivalent. The host cell may or may not naturally express aGHSR on the cell membrane. Preferably, once transformed, the host cellsare able to express the growth hormone secretagogue receptor or afunctional equivalent on the cell membrane. Depending on the host cell,it may be desirable to adapt the DNA so that particular codons are usedin order to optimize expression. Such adaptations are known in the art,and these nucleic acids are also included within the scope of thisinvention. Generally, mammalian cell lines, such as COS, HEK-293, CHO,HeLa, NS/O, CV-1, GC, GH3 or VERO cells are preferred host cells, butother cells and cell lines such as Xenopus oocytes or insect cells, mayalso be used.

Growth hormone secretagogue related receptors are related to GHRS, butare encoded by a distinct gene. The GHRR genes may be identified byhybridization (using relaxed or moderate stringency post-hybridizationalwashing conditions) of cDNA of GHR DNA to genonic DNA. These sequenceshave a high degree of similarity to GHR.

Another aspect of this invention is a process for identifying nucleicacids encoding growth hormone secretagogue related receptors comprisinghybridizing a first nucleic acid encoding a growth hormone secretagoguereceptor with a second nucleic acid suspected of comprising nucleicacids encoding a growth hormone secretagogue, wherein the hybridizingtakes place under relaxed or moderate post hybridizational washingconditions; and identify areas of the second nucleic acid wherehybridization occurred.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the DNA of Swine GHSR (type I) contained in Clone 7-3 (SEQ IDNO:1).

FIG. 2 is the amino acid sequence of swine GHSR encoded by the DNA ofFIG. 1 (SEQ ID NO:2).

FIG. 3 is the entire open reading frame of the type I clone of FIG. 1(SEQ ID NO:3).

FIG. 4 is the DNA of Swine GHSR (type II) contained in Clone 13751 (SEQID NO:4).

FIG. 5 is the amino acid sequence of swine GHSR (type II) encoded by theDNA of FIG. 4 (SEQ ID NO:5).

FIG. 6 is the DNA for human GHSR (Type I) contained in Clone 1146 (SEQID NO:6).

FIG. 7 is the amino acid sequence of human GHSR (type 1) encoded by theDNA of FIG. 6 (SEQ ID NO:7).

FIG. 8 is the entire open reading frame of Type I GHSR, encoded by theDNA sequence of FIG. 6 (SEQ ID NO:8).

FIGS. 9A and 9B are the DNA for human GHSR (type II) contained in Clone1141 (SEQ ID NO:9).

FIG. 10 is the amino acid sequence of human GHSR (Type II) encoded byClone 1141 (SEQ ID NO:10).

FIG. 11 is the DNA for human GHSR (Type I) contained in Clone 1143 (SEQID NO:11).

FIG. 12 is the amino acid sequence of human GHSR (Type I) encoded byClone 1432 (SEQ ID NO:12).

FIGS. 13A and 13B compare to ORF of swine Type I (SEQ ID NO:3; lackingthe MET initiator of the full length GHSR and lacking 12 additionalamino acids) to the homologous domain of swine Type II (SEQ ID NO:5)receptors.

FIGS. 14A and 14B compare the homologous domain of human Type I (SEQ IDNO:8) and Type II (SEQ ID NO:10) receptors (the amino terminal sequencelacks the MET initiator and four additional amino acids).

FIG. 15 compares the ORFs of swine Type I (SEQ ID NO:3) and human Type I(SEQ ID NO:8) receptors (the amino terminal sequence lacks the METinitiator and 12 additional amino acids).

FIGS. 16A and 16B compare full length swine Type II (SEQ ID NO:5) andhuman Type II (SEQ ID NO:10) receptors.

FIG. 17 is a schematic diagram depicting the physical map of swine andhuman growth hormone secretagogue receptor cDNA clones.

FIG. 18 is a graph demonstrating the pharmacology of the expressed swineand human growth hormone secretagogue receptors in Xenopus oocytes usingthe aequorin bioluminescence assay.

FIG. 19 is a table demonstrating the pharmacology of the expressed swineand human growth hormone secretagogue receptors in Xenopus oocytes usingthe aequorin bioluminescence assay and various secretagogues.

FIG. 20 is a graph representing the pharmacology of the pure expressedswine growth hormone secretagogue receptor in COS-7 cells using the³⁵S-labeled Compound A binding assay.

FIG. 21 is a table representing the competition analysis with the pureexpressed swine growth hormone secretagogue receptor in COS-7 cellsusing the ³⁵S-labeled Compound A binding assay and various secretagoguesand other G-protein coupled-receptors (GPC-Receptors) ligands in acompetition assay.

FIG. 22 is the amino acid sequence of the full length human GHSR (typeI) encoded by clone 11304 (SEQ ID NO:13).

FIGS. 23A-D are the rat GHSR DNA sequence from the Met Initiation codonto the Stop codon (SEQ ID NO:14). This sequence includes an intron.

FIGS. 24A and 24B are the open reading frame only of the rat GHSR ofFIG. 23 (SEQ ID NO:15).

FIG. 25 is the deduced amino acid sequence of the ORF of FIG. 24 (SEQ IDNO:16).

FIG. 26 shows the expression of functional rat GHSR in transfectedHEK-293 cells.

As used throughout the specification and claims, the followingdefinitions shall apply:

Growth Hormone Secretagogue—any compound or agent that directly orindirectly stimulates or increases the release of growth hormone in ananimal.

Ligands—any molecule which binds to GHSR of this invention. Theseligands can have either agonist, partial agonist, partial antagonist orantagonist activity.

Free from receptor-associated proteins—the receptor protein is not in amixture or solution with other membrane receptor proteins.

Free from associated nucleic acids—the nucleic acid is not covalentlylinked to DNA which it is naturally covalently linked in the organism'schromosome.

Isolated receptor—the protein is not in a mixture or solution with anyother proteins.

Isolated nucleic acid—the nucleic acid is not in a mixture or solutionwith any other nucleic acid.

Functional equivalent—a receptor which does not have the exact sameamino acid sequence of a naturally occurring growth hormone secretagoguereceptor, due to alternative splicing, deletions, mutations, oradditions, but retains at least 1%, preferably 10%, and more preferably25% of the biological activity of the naturally occurring receptor. Suchderivatives will have a significant homology with a natural GHSR and canbe detected by reduced stringency hybridization with a DNA sequenceobtained from A GHSR. The nucleic acid encoding a functional equivalenthas at least about 50% homology at the nucleotide level to a naturallyoccurring receptor nucleic acid.

Purified receptor—the receptor is at least about 95% pure.

Purified nucleic acid—the nucleic acid is at least about 95% pure.

CompoundA—(N-[1(R)-[(1,2-dihydro-1-methane-sulfonylspiro[3H-indole-3,4′-piperidin]-1′-yl)carbonyl]-2-(phenyl-methyloxy)ethyl]-2-amino-2-methylpropanamide, described in Patchett, 1995 Proc. Natl. Acad. Sci.92:7001-7005.

CompoundB—3-amino-3-methyl-N-(2,3,4,5-tetra-hydro-2-oxo-1-{[2′-1H-tetrazol-5-yl)(1,1′-biphenyl)-4-yl]methyl}-1H-benzazepin-3(R)yl-butanamide,described in Patchett, 1995 Proc. Natl. Acad. Sci. 92:7001-7005.

CompoundC—3-amino-3-methyl-N-(2,3,4,5-tetrahydro-2-oxo-1-{[2′-1H-tetrazol-5-yl)(1,1′-biphenyl)-4-yl]methyl}1H-benzazepin-3(S)yl-butanamide,described in U.S. Pat. No. 5,206,235.

Standard or high stringency post hybridizational washingconditions—6×SSC at 55° C.

Moderate post hybridizational washing conditions—6×SSC at 45° C.

Relaxed post hybridizational washing conditions—6×SSC at 30° C.

The proteins of this invention were found to have structural featureswhich are typical of the 7-transmembrane domain (TM) containingG-protein linked receptor superfamily (GPC-R's or 7-TM receptors). Thusgrowth hormone secretagogue family of receptors make up new members ofthe GPC-R family of receptors. The intact GHSRs of this invention werefound to have the general features of GPC-R's, including seventransmembrane regions, three intra- and extracellular loops, and theGPC-R protein signature sequence. The TM domains and GPC-R proteinsignature sequence are noted in the protein sequences of the Type I GHSreceptor in FIGS. 3 and 8 (SEQ ID NOS:3 and 8 respectively. Not allregions are required for functioning, and therefore this invention alsocomprises functional receptors which lack one or more non-essentialdomains.

The GHSRs of this invention share some sequence homology with previouslycloned GPC-receptors including the rat and human neurotensin receptor(approximately 32% identity) and the rat and human TRH receptor(approximately 30% identity).

The GHSRs of this invention were isolated and characterized usingexpression cloning techniques in Xenopus oocytes. The cloning was madedifficult by three factors. First, prior to this invention, there wasvery little information available about the biochemical characteristicsand intracellular signaling/effector pathways of the proteins. Thus,cloning approaches which depended on the use of protein sequenceinformation for the design of degenerate oligonucleotides to screen cDNAlibraries or utilize PCR could not be effectively utilized. Inaccordance with this invention, therefore, receptor bioactivity neededto be determined.

Secondly, the growth hormone secretagogue receptor does not occur inabundance—it is present on the cell membrane in about 10 fold lessconcentration than most other membrane receptors. In order tosuccessfully clone the receptors in accordance with this invention,exhaustive precautions had be taken to ensure that the GHSR wasrepresented in a cDNA library to be screened. This required isolation ofintact, undegraded and pure poly (A)⁺ mRNA, and optimization of cDNAsynthesis to maximize the production of full-length molecules. Inaddition, a library of larger size than normal needed to be screened(approximately 0.5 to 1×10⁷ clones) to increase the probability that afunctional cDNA clone may be obtained.

Thirdly, no permanent cell line which expresses this receptor is known.Therefore, primary pituitary tissue had to be used as a source for mRNAor protein. This posed an additional obstacle because most primarytissues express lower amounts of a given receptor than an immortalizedcell line that may be maintained in tissue culture or some tumormaterials. Further, the surgical removal of a pig pituitary andextraction of biologically-active intact mRNA for the construction of acDNA expression library is considerably more difficult than theextraction of mRNA from a tissue culture cell line. Along with the needto obtain fresh tissue continuously, there are problems associated withits intrinsic inter-animal and inter-preparation variability. Thedevelopment of cell lines expressing a receptor of this invention istherefore a significant aspect of this invention.

Yet another aspect of this invention is the development of an extremelysensitive, robust, reliable and high-throughput screening assay whichcould be used to identify portions of a cDNA library containing thereceptor. This assay is described and claimed in co-pending patentapplications Serial No. 60/008,584, filed Dec. 13, 1995, and filedherewith.

Briefly, the ability to identify cDNAs which encode growth hormonesecretagogue receptors depended upon two discoveries made in accordancewith this invention: 1) that growth hormone secretagogue receptor-ligandbinding events are transduced through G proteins; and 2) that aparticular G protein subunit, G_(a11), must be present in the cells inorder to detect receptor activity. Only when these two discoveries weremade could an assay be devised to detect the presence of GHSR-encodingDNA sequences.

When the GHSR is bound by ligand (a growth hormone secretagogue), theG-proteins present in the cell activate phosphatidylinositol-specificphospholipase C (PI-PLC), an enzyme which releases intracellularsignaling molecules (diacylglycerol and inositol triphosphate), which inturn start a cascade of biochemical events that promote calciummobilization. This can be used as the basis of an assay. A detectormolecule which can respond to changes in calcium concentrations, such asaequorin, a jellyfish photoprotein, is introduced into a cell along witha complex pool of up to 10,000 individual RNAs from a cDNA expressionlibrary, at least one of which may encode a GHSR. The cell is thenexposed to a known growth hormone secretagogue, such as Compound A orCompound B. If one or more RNAs encodes a GHSR, then the secretagogueligand will bind the receptor, G-protein will be activated, the calciumlevel will fluctuate, and the aequorin will produce measurablebioluminescence. Once a positive result is found, the procedure can berepeated with a sub-division of the RNA pool (for example, approximately1,000, then approximately 500, then approximately 50, and then pureclones) until a single clone is identified from which RNA can begenerated which encodes a GHSR.

Using this general protocol in Xenopus oocytes with a swine cDNAexpression library, Clone 7-3 was identified as containing nucleic acidencoding a swine GHSR. The insert of the cDNA clone is approximately 1.5kb in size, and downstream from the presumed initiator methionine (MET),contains an open reading frame (ORF) encoding 302 amino acids(M_(r)=34,516). The DNA and deduced amino acid sequence are given inFIGS. 1 and 2 (SEQ ID NOS:1 and 2, respectively). When hydropathyanalysis (e.g. Kyte-Doolittle; Eisenberg, Schwartz, Komaron and Wall) isperformed on the protein sequence of clone 7-3, only 6 predictedtransmembrane domains are present downstream of the presumed METinitiator. Translation of the longest ORF encoded in clone 7-3 encodes aprotein of 353 amino acids (M_(r)=39,787); however an apparent METinitiator cannot be identified for this longer reading frame (FIG. 3).This longer reading frame is significant since 7 transmembrane segmentsare encoded in the 353 amino acids protein in which a MET translationinitiation codon located upstream of TM1 is absent. In addition, thislonger protein also shares homology with known G-protein coupledreceptors in its predicted TM1 domain (FIG. 3 and next sections). Thus,clone 7-3 while truncated at its amino terminus, is fully functional,demonstrating that clone 7-3 is but one embodiment of a functionalequivalent of a native GHSR.

The resultant cDNA clone (or shorter portions of, for instance only 15nucleotides long) may be used to probe libraries under hybridizationconditions to find other receptors which are similar enough so that thenucleic acids can hybridize, and is particularly useful for screeninglibraries from other species. Using this procedure, additional human,swine, and rat GHSR cDNAs have been cloned and their nucleotidesequences determined. Further, hybridization of a cDNA to genomic DNAdemonstrated that the Type I receptor (see below) is encoded by a singlegene that is highly conserved. Human, monkey, rat, mouse, dog, cow,chicken and invertebrate DNA all yielded a single hybridizing species athigh stringency post-hybridization conditions. Therefore, this inventionis not limited to any particular species.

A swine pituitary library, a human pituitary library, and a ratpituitary library were hybridized with a radiolabeled cDNA derived fromthe open reading frame of the swine GHSR clone 7-3. 21 positive humanGHSR cDNA clones were isolated and five swine library pools yielded astrong hybridization signal and contained clones with inserts largerthan clone 7-3, as judged by their insert size on Southern blots. Asingle rat cDNA clone was also isolated.

Nucleotide sequence analysis revealed two types of cDNAs for both thehuman and swine GHSR cDNAs. The first (Type I) encodes a proteinrepresented by clone 7-3, encoding seven transmembrane domains. The fulllength open reading frame appears to extend 13 amino acids beyond thelargest predicted open reading frame of clone 7-3 (353 amino acids). Thesecond (type II) diverges in its nucleotide sequence from the type IcDNA at its 3′-end, just after the predicted second amino acid of thesixth transmembrane domain (TM-6).

In the type II cDNAs, TM-6 is truncated and fused to a short contiguousreading frame of only 24 amino acids, followed by a translation stopcodon. Swine clone 1375 is an example of a, Type II cDNA (FIGS. 4 and 5;SEQ ID.NOs:4 and 5, respectively). These 24 amino acids beyond TM-6 arehighly conserved when compared between human and swine cDNAs. The DNAand amino acid sequences of the human GHSR Type I and II are given inFIGS. 6-12; SEQ ID NOs:6-12, respectively. A full length cDNA encodingthe human Type I receptor, that is, a molecule encoding 7-TM domainswith-an initiator MET in a favorable context preceded by an inframetermination codon is isolated, and termed clone 11304. The predicted ORFof clone 11304 for the full length Type I GHSR measures 366 amino acids(M_(r)=41,198; FIG. 22). The full length human Type II cDNA encodes apolypeptide of 289 amino acids (M_(r)=32,156; FIGS. 9A, 9B and 10; SEQID NOs: 9 and 10, respectively.

Sequence alignments performed at both the nucleic acid and proteinlevels show that Type I and II GHSR's are highly related to each otherand across species (FIGS. 13-16). The human and swine GHSR sequences are93% identical and 98% similar at the amino acid level.

The nucleotide sequence encoding the missing amino terminal extension ofswine Type I clone 7-3 is derived from the predicted full length humanType I clone and the human and swine Type II cDNAs. The reading frame ofthe full length clones extended 13 amino acids beyond the amino terminalsequence of clone 7-3 and this sequence was conserved in 12/13 aminoacid residues, when compared between human and swine. The amino terminalextension includes a translation initiator methionine in a favorablecontext according to Kosak's rule, with the reading frame furtherupstream being interrupted by a stop codon. A schematic physical map ofType I and II swine and human cDNA clones is given in FIG. 17.

The rat clone was also further investigated. Sequence analysis revealedthe presence of a non-coding intronic sequence at nt 790 correspondingto a splice-donor site (see FIGS. 23A-D, 24A-B, and 25, respectively).The G/GT splice-donor site occurs two amino acids after the completionof the predicted transmembrane domain 5 (leucine 263), thus dividing therGHSR into an amino-terminal segment (containing the extracellulardomain, TM-1 through TM-5, and the first two intra- and extra-cellularloops) and a carboxy-terminal segment (containing TM-6, TM-7, the thirdintra- and extra-cellular loops, and the intra-cellular domain). Thepoint of insertion and flanking DNA sequence are highly conserved, andalso present in both human and swine Type I and II cDNAs.

Comparison of the complete open reading frame encoding the rat GHSRprotein to human and swine homologs reveals a high degree of sequenceidentity (rat vs. human, 95.1% ; rat vs. swine 93.4%.

The human GHSR can be assigned by fluorescent in situ hybridizationanalysis [FISH; as described in Cytogenet, Cell Genet 69: 196 (1995)] tothe cytogenetic band 3Q26.2. The mouse gene is located on 3A3.

Human and swine Type I cRNAs expressed in oocytes were functional andresponded to Compound A concentrations ranging from 1 mM to as low as0.1 nM in the aequorin bioluminescence assay. Human or swine TypeII-derived cRNAs that are truncated in TM-6 failed to give a responsewhen injected into oocytes and these represent a receptor subtype whichmay bind the GHS, but cannot effectively activate the intracellularsignal transduction pathway. In addition the type II receptor mayinteract with other proteins and thus reconstitute a functional GHSR.Proteins such as these which may have ligand-binding activity, but arenot active in signal transduction are particularly useful forligand-binding assays. In these cases, one may also over-express amutant protein on the cell membrane and test the binding abilities ofputative labeled ligands. By using a non-signaling mutant which isconstitutively in a high affinity state, binding can be measured, but noadverse metabolic consequences would result. Thus non-signaling mutantsare an important aspect of this invention.

The pharmacological characterization of human, Type I swine, Type I andrat receptors in the aequorin bioluminescence assay in oocytes issummarized in FIGS. 18, 19, and 26. Peptidyl and non-peptidyl bioactiveGHS's were active in a similar rank order of potency as observed for thenative pituitary receptor. Independent confirmatory evidence that theType I GHSR (shown for swine clone 7-3) encodes a fully-functional GHSRis given by the finding that when clone 7-3 is expressed transiently inmammalian COS-7 cells, high affinity (K_(D)˜0.2 nM), saturable(B_(max)˜80 fmol/mg protein) and specific binding (>90% displaced by 50nM unlabeled Compound A) is observed for ³⁵S-Compound A (FIGS. 20 and21).

The GHSR receptors of this invention may be identified by hybridizationof a GHSR cDNA to genomic DNA, under relaxed or moderate posthybridizational washing conditions. This analysis yields a discreetnumber of hybridizing bands. A suitable human genomic library which canbe used in this procedure is PAC (as described in Nature Genetics 6:84(1994)) and a suitable mouse genomic library is BAC (as described inProc Natl Acad Sci USA 89: 8794 (1992).

Due to the high degree of homology to GHSRs, the GHSRs of this inventionare believed to function similarly to GHSRs and have similar biologicalactivity. They are useful in understanding the biological andphysiological pathways involved in an organisms growth. They may be alsoused to scan for growth hormone secretagogue agonists and antagonists;as in particular to test the specificity of identified ligands.

Heterotrimeric G proteins, consisting of a, b and g subunits, serve torelay information from cell surface receptors to intracellulareffectors, such as phospholipase C and adenylate cyclase. The G-proteinalpha subunit is an essential component of the intracellular signaltransduction pathway activated by receptor-ligand interaction. In theprocess of ligand-induced GPCR activation, the Ga subunit of a trimericGabg exchanges its bound GDP for GTP and dissociate from the bgheterodimer. The dissociated subunit serves as the active signaltransducer, often in concert with the bg complex, thus starting theactivation of the intracellular signal transduction pathway. Bydefinition, cell surface receptors which couple intracellularly throughG protein interactions are termed GPC-R's. This interaction has mainlybeen characterized with respect to the type of G-alpha (G_(a)) subunitwhich is primarily involved in the signal transduction process. G_(a)subunits are classified into sub-families based on sequence identity andthe main type of effectors to which they are coupled have beencharacterized: G_(s), activate adenylate cyclase; G_(i/o/t), inhibitadenylate cyclase; G_(q/11), activate PI-PLC; and G_(12/13), effectorunknown.

Expression of several receptors in heterologous cells has been shown tobe increased by the co-expression of certain G_(a) subunits. Thisobservation formed the basis for the rationale to the use of G_(a)subunits of several sub-families in conjunction with a source of GHSR(swine poly[A⁺] mRNA) to test if a GHS-induced functional response couldbe measured in the Xenopus oocyte system. GHS-induced responses weredetected and were found to be strictly dependent on G_(a11)co-expression in this system, an unprecedented finding outlining thespecificity of the interaction. Thus another aspect of this invention isa method of detecting a GHS response comprising co-expressing a G_(a11)protein subunit in a cell also expressing a GHSR, exposing the cell to aGHS, and detecting the response.

Ligands detected using assays described herein may be used in thetreatment of conditions which occur when there is a shortage of growthhormone, such as observed in growth hormone deficient children, elderlypatients with musculoskeletal impairment and recovering from hipfracture, and osteoporosis.

The GHSR and fragments are immunogenic. Thus, another aspect of thisinvention is antibodies and antibody fragments which can bind to GHSR ora GHSR fragment. These antibodies may be monoclonal antibodies andproduced using either hybridoma technology or recombinant methods. Theymay be used as part of assay systems or to deduce the function of a GHSRpresent on a cell membrane.

A further aspect of this invention are antisense oligonucleotidesnucleotides which can bind to GHSR nucleotides and modulate receptorfunction or expression.

A further aspect of this invention is a method of increasing the amountof GHSRs on a cell membrane comprising, introducing into the cell anucleic acid encoding a GHSR, and allowing expression of the GHSR.

A GHS receptor, preferably imobilized on a solid support, may be useddiagnostically for the determination of the concentration of growthhormone secretagogues, or metabolites thereof, in physiological fluids,e.g., body fluids, including serum, and tissue extracts, as for examplein patients who are undergoing therapy with a growth hormonesecretagogue.

The administration of a GHS receptor to a patient may also be employedfor purposes of: amplifying the net effect of a growth hormonesecretagogue by providing increased downstream signal followingadministration of the growth hormone secretagogue thereby diminishingthe required dosage of growth hormone secretagogue; or diminishing theeffect of an overdosage of a growth hormone secretagogue during therapy.

The following, non-limiting Examples are presented to better illustratethe invention.

EXAMPLE 1

Oocyte Preparation and Selection

Xenopus laevis oocytes were isolated and injected using standard methodspreviously described by Arena, et al., 1991, Mol. Pharmacol. 40,368-374, which is hereby incorporated by reference. Adult female Xenopuslaevis frogs (purchased from Xenopus One, Ann Arbor, Mich.) wereanesthetized with 0.17% tricaine methanesulfonate and the ovaries weresurgically removed and placed in a 60 mm culture dish (Falcon)containing OR-2 medium without calcium (82.5 mM NaCl, 2 mM KCl, 2.5 mMsodium pyruvate, 1 mM MgCl₂, 100 m/ml penicillin, 1 mg/ml streptomycin,5 mM HEPES, pH=7.5; ND-96 from Specialty Media, N.J.). Ovarian lobeswere broken open, rinsed several times, and oocytes were released fromtheir sacs by collagenase A digestion (Boehringer-Mannheim; 0.2% for 2-3hours at 18° C.) in calcium-free OR-2. When approximately 50% of thefollicular layers were removed, Stage V and VI oocytes were selected andplaced in ND-86 with calcium (86 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 1.8 mMCaCl₂, 2.5 mM sodium pyruvate, 0.5 mM theopylline, 0.1 mM gentamycin, 5mM HEPES [pH=7.5]). For each round of injection, typically 3-5 frogswere pre-tested for their ability to express a control G-protein linkedreceptor (human gonadotropin-releasing hormone receptor) and show arobust phospholipase C intracellular signaling pathway (incubation with1% chicken serum which promotes calcium mobilization by activation ofphospholipase C). Based on these results, 1-2 frogs were chosen forlibrary pool injection (50 nl of cRNA at a concentration of 25 ng(complex pools) to 0.5 ng (pure clone) per oocyte usually 24 to 48 hoursfollowing oocyte isolation.

EXAMPLE 2

mRNA Isolation

Total RNA from swine (50-80 kg, Yorkshire strain) pituitaries(snap-frozen in liquid nitrogen within 1-2 minutes of animal sacrifice)was prepared by a modified phenol:guanidinium thiocyanate procedure(Chomczynski, et al., 1987 Anal. Biochem. 162:156-159, using theTRI-Reagent LS as per the manufacturer's instructions (MolecularResearch Center, Cincinnati, Ohio). Typically, 5 mg of total RNA wasobtained from 3.5 g wet weight of pituitary tissue. Poly (A)⁺ RNA wasisolated from total RNA by column chromatography (two passes) on oligo(dT) cellulose (Pharmacia, Piscataway, N.J.). The yield of poly (A)⁺mRNA from total RNA was usually 0.5%. RNA from other tissues wasisolated similarly.

EXAMPLE 3

cDNA Library Construction

First-strand cDNA was synthesized from poly (A)⁺ mRNA using M-MLV RNAse(−) reverse transcriptase (Superscript, GIBCO-BRL, Gaithersberg, Md.) asper the manufacturer's instructions with an oligo (dT)/Not Iprimer-adapter. Following second-strand cDNA synthesis, double-strandedcDNA was subjected to the following steps: 1) ligation to EcoR Iadapters, 2) Not I digestion, and 3) enrichment for large cDNAs andremoval of excess adapters by gel filtration chromatography on aSephacryl S-500 column (Pharmacia). Fractions corresponding to highmolecular weight cDNA were ligated to EcoR I/Not I digested pSV-7, aeucaryotic expression vector capable of expressing cloned cDNA inmammalian cells by transfection (driven by SV-40 promoter) and inoocytes using in vitro transcripts (initiated from the T7 RNA polymerasepromoter). pSV-7 was constructed by replacing the multiple cloning sitein pSG-5 (Stratagene, La Jolla, Calif.; Green, S. et al., 1988 NucleicAcids Res. 16:369), with an expanded multiple cloning site. Ligatedvector:cDNA was transformed into E.coli strain DH10B (GIBCO-BRL) byelectroporation with a transformation efficiency of 1×10⁶ pfu/10 ngdouble-stranded cDNA. The library contained approximately 3×10⁶independent clones with greater than 95% having inserts with an averagesize approximating 1.65 kb (range 0.8-2.8 kb). Unamplified librarystocks were frozen in glycerol at −70° C. until needed. Aliquots of thelibrary were amplified once prior to screening by a modification of asolid-state method (Kriegler, M. in Gene Transfer and Expression: ALaboratory Manual Stockton Press, NY 1990). Library stocks were titeredon LB plates and then the equivalent of 500-1000 colonies was added to13 ml of 2×YT media containing 0.3% agarose and 100 mg/ml carbenicillinin a 14 ml round-bottom polypropylene tube (Falcon). The bacterialsuspension was chilled in a wet ice bath for 1 hour to solidify thesuspension, and then grown upright at 37° C. for 24 hrs. The resultantbacterial colonies were harvested by centrifugation at 2000×g at RT for10 min, resuspended in 3 ml 2×YT/carbenicillin. Aliquots were taken forfrozen stocks (5%) and plasmid DNA preparation.

EXAMPLE 4

Plasmid DNA Preparation and cRNA Transcription

Plasmid DNA was purified from pellets of solid-state grown bacteria(1000 pools of 500 independent clones each) using the Wizard Miniprepkit according to the manufacturer's instructions (Promega Biotech,Madison, Wis.). The yield of plasmid DNA from a 14 ml solid-stateamplification was 5-10 mg. In preparation for cRNA synthesis, 4 mg ofDNA was digested with Not I, and the subsequent linearized DNA was madeprotein and RNase-free by proteinase K treatment (10 mg for 1 hour at37° C.), followed by two phenol, two chloroform/isoamyl alcoholextractions, and two ethanol precipitations. The DNA was resuspended inapproximately 15 ml of RNase-free water and stored at −70° C. untilneeded. cRNA was synthesized using a kit from Promega Biotech withmodifications. Each 50 ml reaction contained: 5 ml of linearized plasmid(approximately 1 mg), 40 mM Tris-HCl (pH=7.5), 6 mM MgCl₂, 2 mMspermidine, 10 mM NaCl, 10 mM DTT, 0.05 mg/ml bovine serum albumin, 2units/ml RNasin, 800 mM each of ATP, CTP and UTP, 200 mM GTP, 800 mMm7G(5′)ppp(5′)G, 80 units of T7 RNA polymerase, and approximately 20,000cpm of ³²P-CTP as a trace for quantitation of synthesized RNA by TCAprecipitation. The reaction was incubated for 3 hrs. at 30° C.; 20 unitsof RNase-free DNase was added, and the incubation was allowed to proceedfor an additional 15 min. at 37° C. cRNA was purified by two phenol,chloroform/isoamyl alcohol extractions, two ethanol precipitations, andresuspended at a concentration of 500 ng/ml in RNase-free waterimmediately before use.

EXAMPLE 5

Aequorin Bioluminescence Assay (ABA) and Clone Identification

The ABA requires injection of library pool cRNA (25 ng/egg for poolsizes of 500 to 10,000) with aequorin cRNA (2 ng/egg) supplemented withthe G-protein alpha subunit G_(a11) (2 ng/egg). To facilitatestabilization of synthetic transcripts from aequorin and G_(a11)plasmids, the expression vector pCDNA-3 was modified (terned pcDNA-3v2)by insertion (in the Apa I restriction enzyme site of the polylinker) ofa cassette to append a poly (A) tract on all cRNA's which initiate fromthe T7 RNA polymerase promoter. This cassette includes (5′ to 3′): a BglII site, pA (20) and a Sfi I site which can be used for plasmidlinearization. Polymerase chain reaction (PCR) was utilized to generatea DNA fragment corresponding to the open reading frame (ORF) of theaequorin cDNA with an optimized Kosak translational initiation sequence(Inouye, S. et. ul., 1985, Proc. Natl. Acad. Sci. USA 82:3154-3158).This DNA was ligated into pCDNA-3v2 linearized with EcoR I and Kpn I inthe EcoR I/Kpn I site of pCDNA-3v2. G_(a11) cDNA was excised as a ClaI/Not I fragment from the pCMV-5 vector (Woon, C. et. al., 1989 J. Biol.Chem. 264: 5697-93), made blunt with Klenow DNA polymerase and insertedinto the EcoR V site of pcDNA-3v2. cRNA was injected into oocytes usingthe motorized “Nanoject” injector (Drummond Sci. Co., Broomall, Pa.) ina volume of 50 nl. Injection needles were pulled in a single step usinga Flaming/Brown micropipette puller, Model P-87 (Sutter Instrument Co)and the tips were broken using 53×magnification such that an acute anglewas generated with the outside diameter of the needle being <3 mm.Following injection, oocytes were incubated in ND-96 medium, with gentleorbital shaking at 18° C. in the dark. Oocytes were incubated for 24 to48 hours (depending on the experiment and the time required forexpression of the heterologous RNA) before “charging” the expressedaequorin with the essential chromophore coelenterazine. Oocytes were“charged” with coelenterazine by transferring them into 35 mm dishescontaining 3 ml charging medium and incubating for 2-3 hours with gentleorbital shaking in the dark at 18° C. The charging medium contained 10mM coelenterazine (Molecular Probes, Inc., Eugene, Oreg.) and 30 mMreduced glutathione in OR-2 media (no calcium). Oocytes were thenreturned to ND-86 medium with calcium medium described above andincubation continued in the dark with orbital shaking untilbioluminescence measurements were initiated. Measurement of GHSRexpression in oocytes was performed using a Berthold Luminometer LB953(Wallac Inc., Gaithersburg, Md.) connected to a PC running theAutolumat-PC Control software (Wallac Inc., Gaithersburg, Md.). Oocytes(singly or in pairs) were transferred to plastic tubes (75×12 mm,Sarstedt) containing 2.9 ml Ca⁺⁺-free OR-2 medium. Each cRNA pool wastested using a minimum of 3 tubes containing oocytes. Bioluminescencemeasurements were triggered by the injection of 0.1 ml of 30 mM MK-677(1 mM final concentration) and recordings were followed for 2 min. toobserve kinetic responses consistent with an IP₃-mediated response.

Pool S10-20 was prepared from the unfractionated swine pituitary cDNAlibrary and was composed of 10 pools each of 1000 clones. S10-20 gave apositive signal on two luminometer instruments and the component poolswere then individually tested for activity. From the 10 pools of 1000clones, only pool S271 gave a positive response. This pool was made fromtwo pools of 500 clones designated P541 and P542. Again, only one of thepools, P541, gave a positive bioluminescent signal in the presence of 1mM Compound A. At this point, the bacterial titer was determined in theglycerol stock of P541 such that dilutions could be plated onto LB agarplates containing 100 mg/ml carbenicillin to yield approximately 50colonies per plate. A total of 1527 colonies were picked and replicatedfrom 34 plates. The colonies on the original plates were then washedoff, plasmids isolated, cRNA synthesized and injected into oocytes. cRNAprepared from 8 of the 34 plates gave positive signals in oocytes. Twoplates were selected and the individual colonies from these plates weregrown up, plasmid isolated, cRNA prepared and injected into oocytes. Asingle clonal isolate from each plate (designated as clones 7-3 and28-18) gave a positive bioluminescence response to 1 mM Compound A.Clone 7-3 was further characterized.

EXAMPLE 6

Receptor Characterization

DNA sequencing was performed on both strands using an automated AppliedBiosystems instrument (ABI model 373) and manually by the dideoxy chaintermination method using Sequenase II (US Biochemical, Cleveland, Ohio).Database searches (Genbank 88, EMBL 42, Swiss-Prot 31, PIR 40, dEST,Prosite, dbGPCR ), sequence alignments and analysis of the GHSRnucleotide and protein sequences were carried out using the GCG SequenceAnalysis Software Package (Madison, Wis.; pileup, peptide structure andmotif programs), FASTA and BLAST search programs, and the PC/Genesoftware suite from Intelligenetics (San Francisco, Calif.; proteinanalysis programs). Northern blot analysis was conducted using total (20mg/lane) or poly (A)+ mRNA (5-10 mg/lane) prepared as described above.RNA was fractionated on a 1% agarose gel containing 2.2 M formaldehydeand blotted to a nitrocellulose membrane. Southern blots were hybridizedwith a PCR generated probe encompassing the majority of the ORFpredicted by clone 7-3 (nt 291 to 1132). The probe was radiolabeled byrandom-priming with [a]³²P-dCTP to a specific activity of greater than10⁹ dpm/mg. Southern blots were pre-hybridized at 42° C. for 4 hrs. in5×SSC, 5×Denhardt's solution, 250 mg/ml tRNA, 1% glycine, 0.075% SDS, 50mM NaPO₄ (pH 6) and 50% formamide. Hybridizations were carried out at42° C. for 20 hrs. in 5×SSC, 1×Denhardt's solution, 0.1% SDS, 50 mMNaPO₄, and 50% formamide. RNA blots were washed in 2×SSC, 0.2% SDS at42° C. and at −70° C. RNA size markers were 28S and 18S rRNA and invitro transcribed RNA markers (Novagen). Nylon membranes containing EcoRI and Hind III digested genomic DNA from several species (Clontech; 10mg/lane) were hybridized for 24 hrs. at 30° C. in 6×SSPE, 10×Denhardt's,1% SDS, and 50% formamide. Genomic blots were washed twice with roomtemperature 6×SSPE, twice with 55° C. 6×SSPE, and twice with 55° C.4×SSPE. Additional swine GHSR clones from the swine cDNA library(described above) were identified by hybridization to plasmid DNA (inpools of 500 clones each) immobilized to nylon membranes in a slot-blotapparatus (Scheicher and Schuell). Pure clonal isolates weresubsequently identified by colony hybridization. Swine GHSR clones thatextend further in a 5′ direction were identified using 5′ RACEprocedures (Frohman, M. A., 1993 Methods. Enzymol. 218:340-358, which isincorporated by reference) using swine pituitary poly (A)⁺ mRNA astemplate.

EXAMPLE 7

Human GHSR

Human pituitary homologues of the swine GHSR were obtained by screeninga commercially available cDNA library constructed in the vector lambdaZAP II (Stratagene) as per the manufacturer's instructions.Approximately 1.86×10⁶ phages were initially plated and screened using arandom-primer labeled portion of swine clone 7-3 (described above) ashybridization probe. Twenty one positive clones were plaque purified.The inserts from these clones were excised from the bacteriophage intothe phagemid pBluescript II SK- by co-infection with helper phage asdescribed by the manufacturer (Stratagene). Human clones werecharacterized as has been described above for the swine clone.

EXAMPLE 8

Assays

Mammalian cells (COS-7) were transfected with GHSR expression plasmidsusing Lipofectamine (GIBCO-BRL; Hawley-Nelson, P. 1993, Focus 15:73).Transfections were performed in 60 mm dishes on 80% confluent cells(approximately 4×10⁵ cells) with 8 mg of Lipofectamine and 32 mg of GHSRplasmid DNA.

Binding of ³⁵S-Compound A to swine pituitary membranes and crudemembranes prepared from COS-7 cells transfected with GHSR expressionplasmids was conducted. Crude cell membranes from COS-7 transfectantswere prepared on ice, 48 hrs. post-transfection. Each 60 mm dish waswashed twice with 3 ml of PBS, once with 1 ml homogenization buffer (50mM Tris-HCl [pH 7.4], 5 mM MgCl₂, 2.5 mM EDTA, 30 mg/ml bacitracin). 0.5ml of homogenization buffer was added to each dish, cells were removedby scraping and then homogenized using a Polytron device (Brinkmann,Syosset, N.Y.; 3 bursts of 10 sec. at setting 4). The homogenate wasthen centrifuged for 20 min. at 11,000×g at 0° C. and the resultingcrude membrane pellet (chiefly containing cell membranes and nuclei) wasresuspended in homogenization buffer supplemented with 0.06% BSA (0.1ml/60 mm dish) and kept on ice. Binding reactions were performed at 20°C. for 1 hr. in a total volume of 0.5 ml containing: 0.1 ml of membranesuspension, 10 ml of ³⁵S-Compound A (0.05 to 1 nM; specific activityapproximately 900 Ci/ummol), 10 ml of competing drug and 380-390 ml ofhomogenization buffer. Bound radioligand was separated by rapid vacuumfiltration (Brandel 48-well cell harvester) through GF/C filterspretreated for 1 hr. with 0.5% polyethylenimine. After application ofthe membrane suspension to the filter, the filters were washed 3 timeswith 3 ml each of ice cold 50 mM Tris-HCl [pH 7.4], 10 mM MgCl₂, 2.5 mMEDTA and 0.015% Triton X-100, and the bound radioactivity on the filerswas quantitated by scintillation counting. Specific binding (>90% oftotal) is defined as the difference between total binding andnon-specific binding conducted in the presence of 50 nM unlabeledCompound A.

EXAMPLE 9

Preparation of High Specific Activity Radioligand [³⁵S]-Compound A

[³⁵S]-Compound A was prepared from an appropriate precursor,N-[1(R)-[(1,2-dihydrospiro[3H-indole-3,4′-piperidin]-1′-yl)-carbonyl]-2-(phenyl-methyloxy)ethyl]-2-amino-t-butoxycarbonyl-2-methylpropan-amide,using methane [³⁵S]sulfonyl chloride as described in Dean DC, et al.,1995, In: Allen J, Voges R (eds) Synthesis and Applications ofIsotopically Labelled Compounds, John Wiley & Sons, New York, pp.795-801. Purification by semi-preparative HPLC (Zorbax SB-phenyl column,68% MeOH/water, 0.1% TFA, 5 ml/min) was followed by N-t-BOC cleavageusing 15% trifluro-acetic acid in dichloromethane (25° C., 3 hr) to give[methylsulfonyl-³⁵S]Compound A in near quantitative yield. HPLCpurification (Hamilton PRP-1 4.6×250 mm column, linear gradient of50-75% methanol-water with 1 mM HCl over 30 min, 1.3 ml/min) providedthe ligand in >99% radiochemical purity. The structure was establishedby HPLC coelution with unlabeled Compound A and by mass spectralanalysis. The latter method also indicated a specific activity of ˜1000Ci/mmol.

EXAMPLE 10

DNA Encoding a Rat Growth Hormone Secretagogue Receptor (GHSR) Type Ia

Cross-hybridization under reduced stringency was the strategy utilizedto isolate the rat GHSR type Ia. Approximately 10⁶ phage plaques of aonce-amplified rat pituitary cDNA library in lambda gt11 (RL1051b;Clontech, Palo Alto, Calif.) were plated on E. coli strain Y1090r⁻. Theplaques were transferred to maximum-strength Nytran (Schleicher &Schuell, Keene, N.H.) denatured, neutralized and screened with a 1.6 kbEcoRI/NotI fragment containing the entire coding and untranslatedregions of the swine GHSR, clone 7-3. The membranes were incubated at30° C. in prehybridization solution (50% formamide, 2×Denhardts, 5×SSPE,0.1% SDS, 100 mg/ml salmon sperm DNA) for 3 hours followed by overnightincubation in hybridization solution (50% formamide, 2×Denhardts,5×SSPE, 0.1% SDS, 10% dextran sulfate, 100 mg/ml salmon sperm DNA) with1×10⁶ cpm/ml of [³²P]-labeled probe. The probe was labeled with[³²P]dCTP using a random priming kit (Gibco BRL, Gaithersburg, N.D.).After hybridization the blots were washed two times each with 2×SSC,0.1% SDS (at 24° C., then 37° C., and finally 55° C.). A single positiveclone was isolated following three rounds of plaque purification. Phagecontaining the GHSR was eluted from plate plaques with 1×lambda buffer(0.1M NaCl, 0.01M MgSO₄.7H₂O, 35 mM Tris-HCl, pH 7.5) followingovernight growth of approximately 200 pfu/150 mm dish. After a tenminute centrifugation at 10,000×g to remove debris, the phage solutionwas treated with 1 mg/ml RNAse A and DNAse I for thirty minutes at 24°C., followed by precipitation with 20% PEG (8000)/2M NaCl for two hourson ice, and collection by centrifugation at 10,000×g for twenty minutes.Phage DNA was isolated by incubation in 0.1% SDS, 30 mM EDTA, 50 mg/mlproteinase K for one hour at 68° C., with subsequent phenol (threetimes) and chloroform (twice) extraction before isopropanolprecipitation overnight. The GHSR DNA insert (˜6.4 kb) was sub-clonedfrom lambda gt11 into the plasmid vector Litmus 28 (New England Biolabs,Beverly, Mass.). 2 mg of phage DNA was heated to 65° C. for ten minutes,then digested with 100 units BsiWI (New England Biolab, Bevely, Mass.)at 37° C. overnight. A 6.5 kb fragment was gel purified, electroelutedand phenol/chloroform extracted prior to ligation to BsiWI-digestedLitmus 28 vector.

Double-stranded DNA was sequenced on both strands on a ABI 373 automatedsequencer using the ABI PRISM dye termination cycle sequencing readyreaction kit (Perkin Elmer; Foster City, Calif.).

Comparison of the complete ORF encoding the rat GHSR type Ia proteinsequence to human and swine GHSR homologs reveals a high degree ofsequence identity (rat vs. human, 95.1% ; rat vs. swine 93.4% ).

For sequence comparisons and functional expression studies, a contiguousDNA fragment encoding the complete ORF (devoid of intervening sequence)for the rat GHSR type Ia was generated. The PCR was utilized tosynthesize a amino-terminal fragment from Met-1 to Val-260 with EcoRI(5′) and HpaI (3′) restriction sites appended, while a carboxyl-terminalfragment was generated from Lys-261 to Thr-364 with Dra 1 (5′) and Not I(3′) restriction sites appended. The ORF construct was assembled intothe mammalian expression vector pSV7 via a three-way ligation withEcoRi/Not I-digested pSV7, EcoRI/Hpa I-digested NH₂-terminal fragment,and Dra I/Not I-digested C-terminal fragment.

Functional activity of the ORF construct was assessed by transfecting(using lipofectamine; GIBCO/BRL) 5 mg of plasmid DNA into the aequorinexpressing reporter cell line (293-AEQ17) cultured in 60 mm dishes.Following approximately 40 hours of expression the aequorin in the cellswas charged for 2 hours with coelenterazine, the cells were harvested,washed and pelleted by low speed centrifugation into luminometer tubes.Functional activity was determined by measuring Compound A dependentmobilization of intracellular calcium and concomitant calcium inducedaequorin bioluminescence. Shown in FIG. 26 are three replicate samplesexhibiting Compound A-induced luminescent responses.

16 1063 base pairs nucleic acid single linear cDNA 1 CCTCACGCTGCCAGACCTGG GCTGGGACGC TCCCCCTGAA AACGACTCGC TAGTGGAGGA 60 GCTGCTGCCGCTCTTCCCCA CGCCGCTGTT GGCGGGCGTC ACCGCCACCT GCGTGGCGCT 120 CTTCGTGGTGGGTATCGCGG GCAACCTGCT CACGATGCTG GTAGTGTCAC GCTTCCGCGA 180 GATGCGCACCACCACCAACC TCTACCTGTC CAGCATGGCC TTCTCCGACC TACTCATCTT 240 CCTCTGCATGCCCCTCGACC TCTTCCGCCT CTGGCAGTAC CGGCCTTGGA ACCTTGGCAA 300 CCTGCTCTGCAAACTCTTCC AGTTCGTTAG CGAGAGCTGC ACCTACGCCA CAGTGCTCAC 360 CATCACCGCGCTGAGCGTCG AGCGCTACTT CGCCATCTGC TTCCCGCTGC GGGCCAAGGT 420 AGTGGTCACCAAGGGCCGGG TAAAGCTGGT CATCCTGGTC ATCTGGGCCG TGGCCTTCTG 480 CAGCGCCGGGCCCATCTTCG TGCTGGTCGG AGTGGAGCAT GATAACGGCA CTGACCCTCG 540 GGACACCAACGAGTGCCGCG CCACGGAGTT CGCCGTGCGC TCCGGGCTGC TTACCGTCAT 600 GGTCTGGGTGTCCAGTGTCT TCTTCTTCCT GCCTGTCTTC TGCCTCACTG TGCTCTATAG 660 CCTCATCGGCAGGAAGCTCT GGCGGAGGAA GCGCGGCGAG GCGGCGGTGG GCTCCTCGCT 720 CAGGGACCAGAACCACAAAC AAACCGTGAA AATGCTGGCT GTAGTGGTGT TTGCTTTCAT 780 ACTCTGCTGGCTGCCTTTCC ATGTAGGGCG ATATTTATTT TCCAAATCCT TGGAGCCTGG 840 CTCTGTGGAGATTGCTCAGA TCAGCCAATA CTGCAACCTC GTGTCCTTTG TCCTCTTCTA 900 CCTCAGTGCGGCCATCAACC CTATTCTGTA CAACATCATG TCCAAGAAGT ATCGGGTGGC 960 GGTGTTCAAACTGCTGGGAT TTGAGCCCTT CTCACAGAGG AAACTCTCCA CTCTGAAGGA 1020 TGAAAGTTCTCGGGCCTGGA CAGAATCTAG TATTAATACA TGA 1063 302 amino acids amino acidsingle linear protein 2 Met Leu Val Val Ser Arg Phe Arg Glu Met Arg ThrThr Thr Asn Leu 1 5 10 15 Tyr Leu Ser Ser Met Ala Phe Ser Asp Leu LeuIle Phe Leu Cys Met 20 25 30 Pro Leu Asp Leu Phe Arg Leu Trp Gln Tyr ArgPro Trp Asn Leu Gly 35 40 45 Asn Leu Leu Cys Lys Leu Phe Gln Phe Val SerGlu Ser Cys Thr Tyr 50 55 60 Ala Thr Val Leu Thr Ile Thr Ala Leu Ser ValGlu Arg Tyr Phe Ala 65 70 75 80 Ile Cys Phe Pro Leu Arg Ala Lys Val ValVal Thr Lys Gly Arg Val 85 90 95 Lys Leu Val Ile Leu Val Ile Trp Ala ValAla Phe Cys Ser Ala Gly 100 105 110 Pro Ile Phe Val Leu Val Gly Val GluHis Asp Asn Gly Thr Asp Pro 115 120 125 Arg Asp Thr Asn Glu Cys Arg AlaThr Glu Phe Ala Val Arg Ser Gly 130 135 140 Leu Leu Thr Val Met Val TrpVal Ser Ser Val Phe Phe Phe Leu Pro 145 150 155 160 Val Phe Cys Leu ThrVal Leu Tyr Ser Leu Ile Gly Arg Lys Leu Trp 165 170 175 Arg Arg Lys ArgGly Glu Ala Ala Val Gly Ser Ser Leu Arg Asp Gln 180 185 190 Asn His LysGln Thr Val Lys Met Leu Ala Val Val Val Phe Ala Phe 195 200 205 Ile LeuCys Trp Leu Pro Phe His Val Gly Arg Tyr Leu Phe Ser Lys 210 215 220 SerLeu Glu Pro Gly Ser Val Glu Ile Ala Gln Ile Ser Gln Tyr Cys 225 230 235240 Asn Leu Val Ser Phe Val Leu Phe Tyr Leu Ser Ala Ala Ile Asn Pro 245250 255 Ile Leu Tyr Asn Ile Met Ser Lys Lys Tyr Arg Val Ala Val Phe Lys260 265 270 Leu Leu Gly Phe Glu Pro Phe Ser Gln Arg Lys Leu Ser Thr LeuLys 275 280 285 Asp Glu Ser Ser Arg Ala Trp Thr Glu Ser Ser Ile Asn Thr290 295 300 353 amino acids amino acid single linear protein 3 Leu ThrLeu Pro Asp Leu Gly Trp Asp Ala Pro Pro Glu Asn Asp Ser 1 5 10 15 LeuVal Glu Glu Leu Leu Pro Leu Phe Pro Thr Pro Leu Leu Ala Gly 20 25 30 ValThr Ala Thr Cys Val Ala Leu Phe Val Val Gly Ile Ala Gly Asn 35 40 45 LeuLeu Thr Met Leu Val Val Ser Arg Phe Arg Glu Met Arg Thr Thr 50 55 60 ThrAsn Leu Tyr Leu Ser Ser Met Ala Phe Ser Asp Leu Leu Ile Phe 65 70 75 80Leu Cys Met Pro Leu Asp Leu Phe Arg Leu Trp Gln Tyr Arg Pro Trp 85 90 95Asn Leu Gly Asn Leu Leu Cys Lys Leu Phe Gln Phe Val Ser Glu Ser 100 105110 Cys Thr Tyr Ala Thr Val Leu Thr Ile Thr Ala Leu Ser Val Glu Arg 115120 125 Tyr Phe Ala Ile Cys Phe Pro Leu Arg Ala Lys Val Val Val Thr Lys130 135 140 Gly Arg Val Lys Leu Val Ile Leu Val Ile Trp Ala Val Ala PheCys 145 150 155 160 Ser Ala Gly Pro Ile Phe Val Leu Val Gly Val Glu HisAsp Asn Gly 165 170 175 Thr Asp Pro Arg Asp Thr Asn Glu Cys Arg Ala ThrGlu Phe Ala Val 180 185 190 Arg Ser Gly Leu Leu Thr Val Met Val Trp ValSer Ser Val Phe Phe 195 200 205 Phe Leu Pro Val Phe Cys Leu Thr Val LeuTyr Ser Leu Ile Gly Arg 210 215 220 Lys Leu Trp Arg Arg Lys Arg Gly GluAla Ala Val Gly Ser Ser Leu 225 230 235 240 Arg Asp Gln Asn His Lys GlnThr Val Lys Met Leu Ala Val Val Val 245 250 255 Phe Ala Phe Ile Leu CysTrp Leu Pro Phe His Val Gly Arg Tyr Leu 260 265 270 Phe Ser Lys Ser LeuGlu Pro Gly Ser Val Glu Ile Ala Gln Ile Ser 275 280 285 Gln Tyr Cys AsnLeu Val Ser Phe Val Leu Phe Tyr Leu Ser Ala Ala 290 295 300 Ile Asn ProIle Leu Tyr Asn Ile Met Ser Lys Lys Tyr Arg Val Ala 305 310 315 320 ValPhe Lys Leu Leu Gly Phe Glu Pro Phe Ser Gln Arg Lys Leu Ser 325 330 335Thr Leu Lys Asp Glu Ser Ser Arg Ala Trp Thr Glu Ser Ser Ile 340 345 350Asn Thr 1029 base pairs nucleic acid single linear cDNA 4 GCAGCCTCTCACTTCCCTCT TTCCTCTCCT AGCATCCTCC CTGAGAGCCC GCGCTCGATA 60 CTCCTTTGCACTCTTTCGCG CCTAAGAGAA CCTTCTCTGG GACCAGCCGG CTCCACCCTC 120 TCGGTCCTATCCAAGAGCCA GTTAAGCAGA GCCCTAAGCA TGTGGAACGC GACCCCGAGC 180 GAGGAACCGGGGCCCAACCT CACGCTGCCA GACCTGGGCT GGGACGCTCC CCCTGAAAAC 240 GACTCGCTAGTGGAGGAGCT GCTGCCGCTC TTCCCCACGC CGCTGTTGGC GGGCGTCACC 300 GCCACCTGCGTGGCGCTCTT CGTGGTGGGT ATCGCGGGCA ACCTGCTCAC GATGCTGGTA 360 GTGTCACGCTTCCGCGAGAT GCGCACCACC ACCAACCTCT ACCTGTCCAG CATGGCCTTC 420 TCCGAACTACTCATCTTCCT CTGCATGCCC CTCGAACTCT TCCGCCTTTG GCAGTACCGG 480 CCTTGGAACCTTGGCAACCT GCTCTGCAAA CTCTTCCAGT TCGTTAGCGA GAGCTGCACC 540 TACGCCACAGTGCTCACCAT CACCGCGCTG AGCGTCGAGC GCTACTTCGC CATCTGCTTC 600 CCGCTGCGGGCCAAGGTAGT GGTCACCAAG GGCCGGGTAA AGCTGGTCAT CCTGGTCATC 660 TGGGCCGTGGCCTTCTGCAG CGCCGGGCCC ATCTTCGTGC TGGTCGGAGT GGAGCATGAT 720 AACGGCACTGACCCTCGGGA CACCAACGAG TGCCGCGCCA CGGAGTTCGC CGTGCGCTCC 780 GGGCTGCTTACCGTCATGGT CTGGGTGTCC AGTGTCTTCT TCTTCCTGCC TGTCTTCTGC 840 CTCACTGTGCTCTATAGCCT CATCGGCAGG AAGCTCTGGC GGAGGAAGCG CGGCGAGGCG 900 GCGGTGGGCTCCTCGCTCAG GGACCAGAAC CACAAACAAA CCGTGAAAAT GCTGGGTGGG 960 TCTCAATGCGCCCTCGAGCT TTCTCTCCCG GGTCCCCTCC ACTCCTCGTG CCTTTTCTCT 1020 TCTCCCTGA1029 289 amino acids amino acid single linear protein 5 Met Trp Asn AlaThr Pro Ser Glu Glu Pro Gly Pro Asn Leu Thr Leu 1 5 10 15 Pro Asp LeuGly Trp Asp Ala Pro Pro Glu Asn Asp Ser Leu Val Glu 20 25 30 Glu Leu LeuPro Leu Phe Pro Thr Pro Leu Leu Ala Gly Val Thr Ala 35 40 45 Thr Cys ValAla Leu Phe Val Val Gly Ile Ala Gly Asn Leu Leu Thr 50 55 60 Met Leu ValVal Ser Arg Phe Arg Glu Met Arg Thr Thr Thr Asn Leu 65 70 75 80 Tyr LeuSer Ser Met Ala Phe Ser Glu Leu Leu Ile Phe Leu Cys Met 85 90 95 Pro LeuGlu Leu Phe Arg Leu Trp Gln Tyr Arg Pro Trp Asn Leu Gly 100 105 110 AsnLeu Leu Cys Lys Leu Phe Gln Phe Val Ser Glu Ser Cys Thr Tyr 115 120 125Ala Thr Val Leu Thr Ile Thr Ala Leu Ser Val Glu Arg Tyr Phe Ala 130 135140 Ile Cys Phe Pro Leu Arg Ala Lys Val Val Val Thr Lys Gly Arg Val 145150 155 160 Lys Leu Val Ile Leu Val Ile Trp Ala Val Ala Phe Cys Ser AlaGly 165 170 175 Pro Ile Phe Val Leu Val Gly Val Glu His Asp Asn Gly ThrAsp Pro 180 185 190 Arg Asp Thr Asn Glu Cys Arg Ala Thr Glu Phe Ala ValArg Ser Gly 195 200 205 Leu Leu Thr Val Met Val Trp Val Ser Ser Val PhePhe Phe Leu Pro 210 215 220 Val Phe Cys Leu Thr Val Leu Tyr Ser Leu IleGly Arg Lys Leu Trp 225 230 235 240 Arg Arg Lys Arg Gly Glu Ala Ala ValGly Ser Ser Leu Arg Asp Gln 245 250 255 Asn His Lys Gln Thr Val Lys MetLeu Gly Gly Ser Gln Cys Ala Leu 260 265 270 Glu Leu Ser Leu Pro Gly ProLeu His Ser Ser Cys Leu Phe Ser Ser 275 280 285 Pro 1088 base pairsnucleic acid single linear cDNA 6 CGCCCAGCGA AGAGCCGGGG TTCAACCTCACACTGGCCGA CCTGGACTGG GATGCTTCCC 60 CCGGCAACGA CTCGCTGGGC GACGAGCTGCTGCAGCTCTT CCCCGCGCCG CTGCTGGCGG 120 GCGTCACAGC CACCTGCGTG GCACTCTTCGTGGTGGGTAT CGCTGGCAAC CTGCTCACCA 180 TGCTGGTGGT GTCGCGCTTC CGCGAGCTGCGCACCACCAC CAACCTCTAC CTGTCCAGCA 240 TGGCCTTCTC CGATCTGCTC ATCTTCCTCTGCATGCCCCT GGACCTCGTT CGCCTCTGGC 300 AGTACCGGCC CTGGAACTTC GGCGACCTCCTCTGCAAACT CTTCCAATTC GTCAGTGAGA 360 GCTGCACCTA CGCCACGGTG CTCACCATCACAGCGCTGAG CGTCGAGCGC TACTTCGCCA 420 TCTGCTTCCC ACTCCGGGCC AAGGTGGTGGTCACCAAGGG GCGGGTGAAG CTGGTCATCT 480 TCGTCATCTG GGCCGTGGCC TTCTGCAGCGCCGGGCCCAT CTTCGTGCTA GTCGGGGTGG 540 AGCACGAGAA CGGCACCGAC CCTTGGGACACCAACGAGTG CCGCCCCACC GAGTTTGCGG 600 TGCGCTCTGG ACTGCTCACG GTCATGGTGTGGGTGTCCAG CATCTTCTTC TTCCTTCCTG 660 TCTTCTGTCT CACGGTCCTC TACAGTCTCATCGGCAGGAA GCTGTGGCGG AGGAGGCGCG 720 GCGATGCTGT CGTGGGTGCC TCGCTCAGGGACCAGAACCA CAAGCAAACC GTGAAAATGC 780 TGGCTGTAGT GGTGTTTGCC TTCATCCTCTGCTGGCTCCC CTTCCACGTA GGGCGATATT 840 TATTTTCCAA ATCCTTTGAG CCTGGCTCCTTGGAGATTGC TCAGATCAGC CAGTACTGCA 900 ACCTCGTGTC CTTTGTCCTC TTCTACCTCAGTGCTGCCAT CAACCCCATT CTGTACAACA 960 TCATGTCCAA GAAGTACCGG GTGGCAGTGTTCAGACTTCT GGGATTCGAA CCCTTCTCCC 1020 AGAGAAAGCT CTCCACTCTG AAAGATGAAAGTTCTCGGGC CTGGACAGAA TCTAGTATTA 1080 ATACATGA 1088 302 amino acidsamino acid single linear protein 7 Met Leu Val Val Ser Arg Phe Arg GluLeu Arg Thr Thr Thr Asn Leu 1 5 10 15 Tyr Leu Ser Ser Met Ala Phe SerAsp Leu Leu Ile Phe Leu Cys Met 20 25 30 Pro Leu Asp Leu Val Arg Leu TrpGln Tyr Arg Pro Trp Asn Phe Gly 35 40 45 Asp Leu Leu Cys Lys Leu Phe GlnPhe Val Ser Glu Ser Cys Thr Tyr 50 55 60 Ala Thr Val Leu Thr Ile Thr AlaLeu Ser Val Glu Arg Tyr Phe Ala 65 70 75 80 Ile Cys Phe Pro Leu Arg AlaLys Val Val Val Thr Lys Gly Arg Val 85 90 95 Lys Leu Val Ile Phe Val IleTrp Ala Val Ala Phe Cys Ser Ala Gly 100 105 110 Pro Ile Phe Val Leu ValGly Val Glu His Glu Asn Gly Thr Asp Pro 115 120 125 Trp Asp Thr Asn GluCys Arg Pro Thr Glu Phe Ala Val Arg Ser Gly 130 135 140 Leu Leu Thr ValMet Val Trp Val Ser Ser Ile Phe Phe Phe Leu Pro 145 150 155 160 Val PheCys Leu Thr Val Leu Tyr Ser Leu Ile Gly Arg Lys Leu Trp 165 170 175 ArgArg Arg Arg Gly Asp Ala Val Val Gly Ala Ser Leu Arg Asp Gln 180 185 190Asn His Lys Gln Thr Val Lys Met Leu Ala Val Val Val Phe Ala Phe 195 200205 Ile Leu Cys Trp Leu Pro Phe His Val Gly Arg Tyr Leu Phe Ser Lys 210215 220 Ser Phe Glu Pro Gly Ser Leu Glu Ile Ala Gln Ile Ser Gln Tyr Cys225 230 235 240 Asn Leu Val Ser Phe Val Leu Phe Tyr Leu Ser Ala Ala IleAsn Pro 245 250 255 Ile Leu Tyr Asn Ile Met Ser Lys Lys Tyr Arg Val AlaVal Phe Arg 260 265 270 Leu Leu Gly Phe Glu Pro Phe Ser Gln Arg Lys LeuSer Thr Leu Lys 275 280 285 Asp Glu Ser Ser Arg Ala Trp Thr Glu Ser SerIle Asn Thr 290 295 300 361 amino acids amino acid single linear protein8 Pro Ser Glu Glu Pro Gly Phe Asn Leu Thr Leu Ala Asp Leu Asp Trp 1 5 1015 Asp Ala Ser Pro Gly Asn Asp Ser Leu Gly Asp Glu Leu Leu Gln Leu 20 2530 Phe Pro Ala Pro Leu Leu Ala Gly Val Thr Ala Thr Cys Val Ala Leu 35 4045 Phe Val Val Gly Ile Ala Gly Asn Leu Leu Thr Met Leu Val Val Ser 50 5560 Arg Phe Arg Glu Leu Arg Thr Thr Thr Asn Leu Tyr Leu Ser Ser Met 65 7075 80 Ala Phe Ser Asp Leu Leu Ile Phe Leu Cys Met Pro Leu Asp Leu Val 8590 95 Arg Leu Trp Gln Tyr Arg Pro Trp Asn Phe Gly Asp Leu Leu Cys Lys100 105 110 Leu Phe Gln Phe Val Ser Glu Ser Cys Thr Tyr Ala Thr Val LeuThr 115 120 125 Ile Thr Ala Leu Ser Val Glu Arg Tyr Phe Ala Ile Cys PhePro Leu 130 135 140 Arg Ala Lys Val Val Val Thr Lys Gly Arg Val Lys LeuVal Ile Phe 145 150 155 160 Val Ile Trp Ala Val Ala Phe Cys Ser Ala GlyPro Ile Phe Val Leu 165 170 175 Val Gly Val Glu His Glu Asn Gly Thr AspPro Trp Asp Thr Asn Glu 180 185 190 Cys Arg Pro Thr Glu Phe Ala Val ArgSer Gly Leu Leu Thr Val Met 195 200 205 Val Trp Val Ser Ser Ile Phe PhePhe Leu Pro Val Phe Cys Leu Thr 210 215 220 Val Leu Tyr Ser Leu Ile GlyArg Lys Leu Trp Arg Arg Arg Arg Gly 225 230 235 240 Asp Ala Val Val GlyAla Ser Leu Arg Asp Gln Asn His Lys Gln Thr 245 250 255 Val Lys Met LeuAla Val Val Val Phe Ala Phe Ile Leu Cys Trp Leu 260 265 270 Pro Phe HisVal Gly Arg Tyr Leu Phe Ser Lys Ser Phe Glu Pro Gly 275 280 285 Ser LeuGlu Ile Ala Gln Ile Ser Gln Tyr Cys Asn Leu Val Ser Phe 290 295 300 ValLeu Phe Tyr Leu Ser Ala Ala Ile Asn Pro Ile Leu Tyr Asn Ile 305 310 315320 Met Ser Lys Lys Tyr Arg Val Ala Val Phe Arg Leu Leu Gly Phe Glu 325330 335 Pro Phe Ser Gln Arg Lys Leu Ser Thr Leu Lys Asp Glu Ser Ser Arg340 345 350 Ala Trp Thr Glu Ser Ser Ile Asn Thr 355 360 1122 base pairsnucleic acid single linear cDNA 9 GCGCCTCACG CTCCCGCTTC GCGGCGCCTGGTCCCTGCGG TCCCCACTCG CTGCGACGCT 60 TTGGGAAGTG CGAGATGGAA CTGGATCGAGAACGCAAATG CGAGGCAGGG CTGGTGACAG 120 CATCCTCCCT ACGCGTCTGC ACCCGCTCCTCCCTCGCACC CTCCCGCGCC TAAGCGGACC 180 TCCTCGGGAG CCAGCTCGGT CCAGCCTCCCAGCGCAGTCA CGTCCCAGAG CCTGTTCAGC 240 TGAGCCGGCA GCATGTGGAA CGCGACGCCCAGCGAAGAGC CGGGGTTCAA CCTCACACTG 300 GCCGACCTGG ACTGGGATGC TTCCCCCGGCAACGACTCGC TGGGCGACGA GCTGCTGCAG 360 CTCTTCCCCG CGCCGCTGCT GGCGGGCGTCACAGCCACCT GCGTGGCACT CTTCGTGGTG 420 GGTATCGCTG GCAACCTGCT CACCATGCTGGTGGTGTCGC GCTTCCGCGA GCTGCGCACC 480 ACCACCAACC TCTACCTGTC CAGCATGGCCTTCTCCGATC TGCTCATCTT CCTCTGCATG 540 CCCCTGGACC TCGTTCGCCT CTGGCAGTACCGGCCCTGGA ACTTCGGCGA CCTCCTCTGC 600 AAACTCTTCC AATTCGTCAG TGAGAGCTGCACCTACGCCA CGGTGCTCAC CATCACAGCG 660 CTGAGCGTCG AGCGCTACTT CGCCATCTGCTTCCCACTCC GGGCCAAGGT GGTGGTCACC 720 AAGGGGCGGG TGAAGCTGGT CATCTTCGTCATCTGGGCCG TGGCCTTCTG CAGCGCCGGG 780 CCCATCTTCG TGCTAGTCGG GGTGGAGCACGAGAACGGCA CCGACCCTTG GGACACCAAC 840 GAGTGCCGCC CCACCGAGTT TGCGGTGCGCTCTGGACTGC TCACGGTCAT GGTGTGGGTG 900 TCCAGCATCT TCTTCTTCCT TCCTGTCTTCTGTCTCACGG TCCTCTACAG TCTCATCGGC 960 AGGAAGCTGT GGCGGAGGAG GCGCGGCGATGCTGTCGTGG GTGCCTCGCT CAGGGACCAG 1020 AACCACAAGC AAACCGTGAA AATGCTGGGTGGGTCTCAGC GCGCGCTCAG GCTTTCTCTC 1080 GCGGGTCCTA TCCTCTCCCT GTGCCTTCTCCCTTCTCTCT GA 1122 289 amino acids amino acid single linear protein 10Met Trp Asn Ala Thr Pro Ser Glu Glu Pro Gly Phe Asn Leu Thr Leu 1 5 1015 Ala Asp Leu Asp Trp Asp Ala Ser Pro Gly Asn Asp Ser Leu Gly Asp 20 2530 Glu Leu Leu Gln Leu Phe Pro Ala Pro Leu Leu Ala Gly Val Thr Ala 35 4045 Thr Cys Val Ala Leu Phe Val Val Gly Ile Ala Gly Asn Leu Leu Thr 50 5560 Met Leu Val Val Ser Arg Phe Arg Glu Leu Arg Thr Thr Thr Asn Leu 65 7075 80 Tyr Leu Ser Ser Met Ala Phe Ser Asp Leu Leu Ile Phe Leu Cys Met 8590 95 Pro Leu Asp Leu Val Arg Leu Trp Gln Tyr Arg Pro Trp Asn Phe Gly100 105 110 Asp Leu Leu Cys Lys Leu Phe Gln Phe Val Ser Glu Ser Cys ThrTyr 115 120 125 Ala Thr Val Leu Thr Ile Thr Ala Leu Ser Val Glu Arg TyrPhe Ala 130 135 140 Ile Cys Phe Pro Leu Arg Ala Lys Val Val Val Thr LysGly Arg Val 145 150 155 160 Lys Leu Val Ile Phe Val Ile Trp Ala Val AlaPhe Cys Ser Ala Gly 165 170 175 Pro Ile Phe Val Leu Val Gly Val Glu HisGlu Asn Gly Thr Asp Pro 180 185 190 Trp Asp Thr Asn Glu Cys Arg Pro ThrGlu Phe Ala Val Arg Ser Gly 195 200 205 Leu Leu Thr Val Met Val Trp ValSer Ser Ile Phe Phe Phe Leu Pro 210 215 220 Val Phe Cys Leu Thr Val LeuTyr Ser Leu Ile Gly Arg Lys Leu Trp 225 230 235 240 Arg Arg Arg Arg GlyAsp Ala Val Val Gly Ala Ser Leu Arg Asp Gln 245 250 255 Asn His Lys GlnThr Val Lys Met Leu Gly Gly Ser Gln Arg Ala Leu 260 265 270 Arg Leu SerLeu Ala Gly Pro Ile Leu Ser Leu Cys Leu Leu Pro Ser 275 280 285 Leu 836base pairs nucleic acid single linear cDNA 11 ATCTGCTCAT CTTCCTCTGCATGCCCCTGG ACCTCGTTCG CCTCTGGCAG TACCGGCCCT 60 GGAACTTCGG CGACCTCCTCTGCAAACTCT TCCAATTCGT CAGTGAGAGC TGCACCTACG 120 CCACGGTGCT CACCATCACAGCGCTGAGCG TCGAGCGCTA CTTCGCCATC TGCTTCCCAC 180 TCCGGGCCAA GGTGGTGGTCACCAAGGGGC GGGTGAAGCT GGTCATCTTC GTCATCTGGG 240 CCGTGGCCTT CTGCAGCGCCGGGCCCATCT TCGTGCTAGT CGGGGTGGAG CACGAGAACG 300 GCACCGACCC TTGGGACACCAACGAGTGCC GCCCCACCGA GTTTGCGGTG CGCTCTGGAC 360 TGCTCACGGT CATGGTGTGGGTGTCCAGCA TCTTCTTCTT CCTTCCTGTC TTCTGTCTCA 420 CGGTCCTCTA CAGTCTCATCGGCAGGAAGC TGTGGCGGAG GAGGCGCGGC GATGCTGTCG 480 TGGGTGCCTC GCTCAGGGACCAGAACCACA AGCAAACCGT GAAAATGCTG GCTGTAGTGG 540 TGTTTGCCTT CATCCTCTGCTGGCTCCCCT TCCACGTAGG GCGATATTTA TTTTCCAAAT 600 CCTTTGAGCC TGGCTCCTTGGAGATTGCTC AGATCAGCCA GTACTGCAAC CTCGTGTCCT 660 TTGTCCTCTT CTACCTCAGTGCTGCCATCA ACCCCATTCT GTACAACATC ATGTCCAAGA 720 AGTACCGGGT GGCAGTGTTCAGACTTCTGG GATTCGAACC CTTCTCCCAG AGAAAGCTCT 780 CCACTCTGAA AGATGAAAGTTCTCGGGCCT GGACAGAATC TAGTATTAAT ACATGA 836 271 amino acids amino acidsingle linear protein 12 Met Pro Leu Asp Leu Val Arg Leu Trp Gln Tyr ArgPro Trp Asn Phe 1 5 10 15 Gly Asp Leu Leu Cys Lys Leu Phe Gln Phe ValSer Glu Ser Cys Thr 20 25 30 Tyr Ala Thr Val Leu Thr Ile Thr Ala Leu SerVal Glu Arg Tyr Phe 35 40 45 Ala Ile Cys Phe Pro Leu Arg Ala Lys Val ValVal Thr Lys Gly Arg 50 55 60 Val Lys Leu Val Ile Phe Val Ile Trp Ala ValAla Phe Cys Ser Ala 65 70 75 80 Gly Pro Ile Phe Val Leu Val Gly Val GluHis Glu Asn Gly Thr Asp 85 90 95 Pro Trp Asp Thr Asn Glu Cys Arg Pro ThrGlu Phe Ala Val Arg Ser 100 105 110 Gly Leu Leu Thr Val Met Val Trp ValSer Ser Ile Phe Phe Phe Leu 115 120 125 Pro Val Phe Cys Leu Thr Val LeuTyr Ser Leu Ile Gly Arg Lys Leu 130 135 140 Trp Arg Arg Arg Arg Gly AspAla Val Val Gly Ala Ser Leu Arg Asp 145 150 155 160 Gln Asn His Lys GlnThr Val Lys Met Leu Ala Val Val Val Phe Ala 165 170 175 Phe Ile Leu CysTrp Leu Pro Phe His Val Gly Arg Tyr Leu Phe Ser 180 185 190 Lys Ser PheGlu Pro Gly Ser Leu Glu Ile Ala Gln Ile Ser Gln Tyr 195 200 205 Cys AsnLeu Val Ser Phe Val Leu Phe Tyr Leu Ser Ala Ala Ile Asn 210 215 220 ProIle Leu Tyr Asn Ile Met Ser Lys Lys Tyr Arg Val Ala Val Phe 225 230 235240 Arg Leu Leu Gly Phe Glu Pro Phe Ser Gln Arg Lys Leu Ser Thr Leu 245250 255 Lys Asp Glu Ser Ser Arg Ala Trp Thr Glu Ser Ser Ile Asn Thr 260265 270 366 amino acids amino acid single linear protein 13 Met Trp AsnAla Thr Pro Ser Glu Glu Pro Gly Phe Asn Leu Thr Leu 1 5 10 15 Ala AspLeu Asp Trp Asp Ala Ser Pro Gly Asn Asp Ser Leu Gly Asp 20 25 30 Glu LeuLeu Gln Leu Phe Pro Ala Pro Leu Leu Ala Gly Val Thr Ala 35 40 45 Thr CysVal Ala Leu Phe Val Val Gly Ile Ala Gly Asn Leu Leu Thr 50 55 60 Met LeuVal Val Ser Arg Phe Arg Glu Leu Arg Thr Thr Thr Asn Leu 65 70 75 80 TyrLeu Ser Ser Met Ala Phe Ser Asp Leu Leu Ile Phe Leu Cys Met 85 90 95 ProLeu Asp Leu Val Arg Leu Trp Gln Tyr Arg Pro Trp Asn Phe Gly 100 105 110Asp Leu Leu Cys Lys Leu Phe Gln Phe Val Ser Glu Ser Cys Thr Tyr 115 120125 Ala Thr Val Leu Thr Ile Thr Ala Leu Ser Val Glu Arg Tyr Phe Ala 130135 140 Ile Cys Phe Pro Leu Arg Ala Lys Val Val Val Thr Lys Gly Arg Val145 150 155 160 Lys Leu Val Ile Phe Val Ile Trp Ala Val Ala Phe Cys SerAla Gly 165 170 175 Pro Ile Phe Val Leu Val Gly Val Glu His Glu Asn GlyThr Asp Pro 180 185 190 Trp Asp Thr Asn Glu Cys Arg Pro Thr Glu Phe AlaVal Arg Ser Gly 195 200 205 Leu Leu Thr Val Met Val Trp Val Ser Ser IlePhe Phe Phe Leu Pro 210 215 220 Val Phe Cys Leu Thr Val Leu Tyr Ser LeuIle Gly Arg Lys Leu Trp 225 230 235 240 Arg Arg Arg Arg Gly Asp Ala ValVal Gly Ala Ser Leu Arg Asp Gln 245 250 255 Asn His Lys Gln Thr Val LysMet Leu Ala Val Val Val Phe Ala Phe 260 265 270 Ile Leu Cys Trp Leu ProPhe His Val Gly Arg Tyr Leu Phe Ser Lys 275 280 285 Ser Phe Glu Pro GlySer Leu Glu Ile Ala Gln Ile Ser Gln Tyr Cys 290 295 300 Asn Leu Val SerPhe Val Leu Phe Tyr Leu Ser Ala Ala Ile Asn Pro 305 310 315 320 Ile LeuTyr Asn Ile Met Ser Lys Lys Tyr Arg Val Ala Val Phe Arg 325 330 335 LeuLeu Gly Phe Glu Pro Phe Ser Gln Arg Lys Leu Ser Thr Leu Lys 340 345 350Asp Glu Ser Ser Arg Ala Trp Thr Glu Ser Ser Ile Asn Thr 355 360 365 3129base pairs nucleic acid single linear cDNA 14 ATGTGGAACG CGACCCCCAGCGAGGAGCCG GAGCCTAACG TCACGTTGGA CCTGGATTGG 60 GACGCTTCCC CCGGCAACGACTCACTGCCT GACGAACTGC TGCCGCTGTT CCCCGCTCCG 120 CTGCTGGCAG GCGTCACCGCCACCTGCGTG GCGCTCTTCG TGGTGGGCAT CTCAGGCAAC 180 CTGCTCACTA TGCTGGTGGTGTCCCGCTTC CGCGAGCTGC GCACCACCAC CAACCTCTAC 240 CTGTCCAGCA TGGCCTTCTCGGATCTGCTC ATCTTCCTGT GCATGCCGCT GGACCTCGTC 300 CGCCTCTGGC AGTACCGGCCCTGGAACTTC GGCGACCTGC TCTGCAAACT CTTCCAGTTT 360 GTCAGCGAGA GCTGCACCTACGCCACGGTC CTCACCATCA CCGCGCTGAG CGTCGAGCGC 420 TACTTCGCCA TCTGCTTCCCTCTGCGGGCC AAGGTGGTGG TCACTAAGGG CCGCGTGAAG 480 CTGGTCATCC TTGTCATCTGGGCCGTGGCT TTCTGCAGCG CGGGGCCCAT CTTCGTGCTG 540 GTGGGCGTGG AGCACGAAAACGGCACAGAT CCCCGGGACA CCAACGAATG CCGCGCCACC 600 GAGTTCGCTG TGCGCTCTGGGCTGCTCACC GTCATGGTGT GGGTGTCCAG CGTCTTCTTC 660 TTTCTACCGG TCTTCTGCCTCACTGTGCTC TACAGTCTCA TCGGGAGGAA GCTATGGCGG 720 AGACGCGGAG ATGCAGCGGTGGGCGCCTCG CTCCGGGACC AGAACCACAA GCAGACAGTG 780 AAGATGCTTG GTGAGTCCTGGCACCCGCTG ACCTTTCTTC CCCCACTGCC TGCCCTTCCC 840 CAGCGGCCTC TATTTCTGTTTCTCATCATC TCCGCTCCCC AAGTCTCTCA AGTCTCTGTC 900 TTTCTCTGCC TCTCTCACCTTGGTTCTCGG TCTCACTGCT TTCTGTTTTC TTCCTGTCTT 960 TTCCTGTATC TTGTCCACGAAAAAGAACCC TCATATTGGT AATTCCTTAA AACGAGGAAC 1020 CTTGGTCTGG GAAAATTGGTCCAAGATGGA AATACCTCAC GGTTTATTGA GCCCCTAATT 1080 GTTAACGGTT TAGCTTCTTGTCTCACATAG AATTTGTGGT TATCAAAGTA ATAATATTAA 1140 GGTAAGCAGG CAGGTAATGGGTTTAGAAAT CACTCCATGG TAAGTCTAAC CACAAATTTG 1200 GGTCACTCTG TTAAGGACGGCTTATAGATG TATTTTGTTT GTTTTCAATA TTGGGATTTG 1260 TTTTCTGCCC TGCATCTTTCTCAGATAATT ACATCCACTC TGTTTAGTCT ATGGTTTTGC 1320 CAGGAGGGGC TTCATGCTGGGGTCTCCTTT TTCTTGTTTT TGTATTTGTC TCCCCAGTAA 1380 TATAGGCCAG GATAGGGTGGAGAAGTCATC CTTTCCTCAA ACTGTCCTTC AGGAAGGTCT 1440 GGGTACTGAA CGGTTACTGCATAAACTCTG CTTCCCCAAA GGCATGTGCT TGGTGTGGTA 1500 AAGTCATGAA GATGGTGCTCATGTCCAAGA GGAACCTCTG ATCTCACTTT TCAAGGGATT 1560 TCATGTTTGC TGACATTTAATACTTGTTAG TTTTTGCAGG GGGATGATTT CTCATTTGCA 1620 ATTTTATTAT TCTCAAATTCTGCATGTCAG AATGTTAGAG ATTTCTCAGG GATGTCAGGT 1680 TCTGTTTCCA GATGAGTGATTGCCCTGTGT CCTCCATTGG ACTGTAAACT CATATGCACC 1740 AGACAGGGTC TACATTGCTGCCGTGGTGCA TAGCCTTCCA TGTGTCACTT AGTCCTAAAG 1800 AGAAGTTACT AATAACCTAATCTCACTAAT CTCACTGGCA TCTCAATGCC GATCCCATTG 1860 TCATCTGAAA ATTTGAAGGGGACATTAAAG TGGCACAGGG ACCAGAACAA TATTTTTCTC 1920 TCATTGCTGA ATTTTAAAAACAATCTAAAA AATTGGAATT CTTGAAGAAA CTATCTTATA 1980 TGACTAAAAT GAAGCCTTGGGTGGGTGCTA ATTATTATTG TCTGGCTTAC CTGCCCCCCC 2040 CACTACTTAT ATCTTTTAGAGATGACACAG ACTTGCTTTC CCTGTGGCTA CTAATCCCAA 2100 TTGCACATTC AGTCCCTTGATAGACTTACT CTAAAAATCT AAGTTCAGCG GTCCACGAAA 2160 CATAACAAAG CCTGTCCTAAAACAGAAAGA AAGAAAGAAA GAAAGAAAGA AAGAAAGAAA 2220 GAAAGAAAGA AAGAAAGAAAACAGAAGACA AACAAGGTCT TTCCCCATTC CCTAACATAC 2280 AGGAATGGAA ATTATTAAGTCTACGTGATA GCCAATGAAT CTGTTTCTTA AGTATGCCCA 2340 CAAGGGTGCT GCCGGAGCCATTGCTCAGGG CTGGAGTATT TACTGGGCAT GCTTGACCCC 2400 AGCATGGAGG GTGAGAAGTGCTCCTGGGAA CTCTGATCCA CTGCTGTGGT GGAGAGCAAA 2460 CACCTGGCCT CATTTATACTTGTTGTCTGT ATAATGCATA TAAATGGGGG ATAATCATTA 2520 CTAAACTGTT TAGCTGAGCCTCATGTCAGT CAATCACAAA GCAGAGTAAT TACCACACAG 2580 ACTGGGAAGC TCAGTGAAGATTGTTAGCGG TTGGTCTGAC AGTCTTGCTG TGTGCTATAG 2640 TGTTAGACCC AACGGAGGCAGTATTTATAA GGAGGGCAGG GTTCCATGTT TCCCGTGTTA 2700 AAGAGCAAGA GATGATGTTTGTCAGTAGGC ATGCAGCTCA TGGTGAAAAG AAAGTCCAGA 2760 CTTAAAGATG TGAAGTGATTTGTGCTTTGC CCCACCCTGA CAGTCTCTCT CTGTGTGCCT 2820 TCAGCTGTGG TGGTGTTTGCTTTCATCCTC TGCTGGCTGC CCTTCCACGT GGGAAGATAC 2880 CTCTTTTCCA AGTCCTTCGAGCCTGGCTCT CTGGAGATCG CTCAGATCAG CCAGTACTGC 2940 AACCTGGTGT CCTTTGTCCTCTTCTACCTC AGCGCTGCCA TCAACCCCAT TCTGTACAAC 3000 ATCATGTCCA AGAAGTACCGGGTGGCAGTG TTCAAACTGC TAGGATTTGA ATCCTTCTCC 3060 CAGAGAAAGC TTTCCACTCTGAAGGATGAG AGTTCCCGGG CCTGGACAAA GTCGAGCATC 3120 AACACATGA 3129 1092base pairs nucleic acid single linear cDNA 15 ATGTGGAACG CGACCCCCAGCGAGGAGCCG GAGCCTAACG TCACGTTGGA CCTGGATTGG 60 GACGCTTCCC CCGGCAACGACTCACTGCCT GACGAACTGC TGCCGCTGTT CCCCGCTCCG 120 CTGCTGGCAG GCGTCACCGCCACCTGCGTG GCGCTCTTCG TGGTGGGCAT CTCAGGCAAC 180 CTGCTCACTA TGCTGGTGGTGTCCCGCTTC CGCGAGCTGC GCACCACCAC CAACCTCTAC 240 CTGTCCAGCA TGGCCTTCTCGGATCTGCTC ATCTTCCTGT GCATGCCGCT GGACCTCGTC 300 CGCCTCTGGC AGTACCGGCCCTGGAACTTC GGCGACCTGC TCTGCAAACT CTTCCAGTTT 360 GTCAGCGAGA GCTGCACCTACGCCACGGTC CTCACCATCA CCGCGCTGAG CGTCGAGCGC 420 TACTTCGCCA TCTGCTTCCCTCTGCGGGCC AAGGTGGTGG TCACTAAGGG CCGCGTGAAG 480 CTGGTCATCC TTGTCATCTGGGCCGTGGCT TTCTGCAGCG CGGGGCCCAT CTTCGTGCTG 540 GTGGGCGTGG AGCACGAAAACGGCACAGAT CCCCGGGACA CCAACGAATG CCGCGCCACC 600 GAGTTCGCTG TGCGCTCTGGGCTGCTCACC GTCATGGTGT GGGTGTCCAG CGTCTTCTTC 660 TTTCTACCGG TCTTCTGCCTCACTGTGCTC TACAGTCTCA TCGGGAGGAA GCTATGGCGG 720 AGACGCGGAG ATGCAGCGGTGGGCGCCTCG CTCCGGGACC AGAACCACAA GCAGACAGTG 780 AAGATGCTTG CTGTGGTGGTGTTTGCTTTC ATCCTCTGCT GGCTGCCCTT CCACGTGGGA 840 AGATACCTCT TTTCCAAGTCCTTCGAGCCT GGCTCTCTGG AGATCGCTCA GATCAGCCAG 900 TACTGCAACC TGGTGTCCTTTGTCCTCTTC TACCTCAGCG CTGCCATCAA CCCCATTCTG 960 TACAACATCA TGTCCAAGAAGTACCGGGTG GCAGTGTTCA AACTGCTAGG ATTTGAATCC 1020 TTCTCCCAGA GAAAGCTTTCCACTCTGAAG GATGAGAGTT CCCGGGCCTG GACAAAGTCG 1080 AGCATCAACA CA 1092 364amino acids amino acid single linear protein 16 Met Trp Asn Ala Thr ProSer Glu Glu Pro Glu Pro Asn Val Thr Leu 1 5 10 15 Asp Leu Asp Trp AspAla Ser Pro Gly Asn Asp Ser Leu Pro Asp Glu 20 25 30 Leu Leu Pro Leu PhePro Ala Pro Leu Leu Ala Gly Val Thr Ala Thr 35 40 45 Cys Val Ala Leu PheVal Val Gly Ile Ser Gly Asn Leu Leu Thr Met 50 55 60 Leu Val Val Ser ArgPhe Arg Glu Leu Arg Thr Thr Thr Asn Leu Tyr 65 70 75 80 Leu Ser Ser MetAla Phe Ser Asp Leu Leu Ile Phe Leu Cys Met Pro 85 90 95 Leu Asp Leu ValArg Leu Trp Gln Tyr Arg Pro Trp Asn Phe Gly Asp 100 105 110 Leu Leu CysLys Leu Phe Gln Phe Val Ser Glu Ser Cys Thr Tyr Ala 115 120 125 Thr ValLeu Thr Ile Thr Ala Leu Ser Val Glu Arg Tyr Phe Ala Ile 130 135 140 CysPhe Pro Leu Arg Ala Lys Val Val Val Thr Lys Gly Arg Val Lys 145 150 155160 Leu Val Ile Leu Val Ile Trp Ala Val Ala Phe Cys Ser Ala Gly Pro 165170 175 Ile Phe Val Leu Val Gly Val Glu His Glu Asn Gly Thr Asp Pro Arg180 185 190 Asp Thr Asn Glu Cys Arg Ala Thr Glu Phe Ala Val Arg Ser GlyLeu 195 200 205 Leu Thr Val Met Val Trp Val Ser Ser Val Phe Phe Phe LeuPro Val 210 215 220 Phe Cys Leu Thr Val Leu Tyr Ser Leu Ile Gly Arg LysLeu Trp Arg 225 230 235 240 Arg Arg Gly Asp Ala Ala Val Gly Ala Ser LeuArg Asp Gln Asn His 245 250 255 Lys Gln Thr Val Lys Met Leu Ala Val ValVal Phe Ala Phe Ile Leu 260 265 270 Cys Trp Leu Pro Phe His Val Gly ArgTyr Leu Phe Ser Lys Ser Phe 275 280 285 Glu Pro Gly Ser Leu Glu Ile AlaGln Ile Ser Gln Tyr Cys Asn Leu 290 295 300 Val Ser Phe Val Leu Phe TyrLeu Ser Ala Ala Ile Asn Pro Ile Leu 305 310 315 320 Tyr Asn Ile Met SerLys Lys Tyr Arg Val Ala Val Phe Lys Leu Leu 325 330 335 Gly Phe Glu SerPhe Ser Gln Arg Lys Leu Ser Thr Leu Lys Asp Glu 340 345 350 Ser Ser ArgAla Trp Thr Lys Ser Ser Ile Asn Thr 355 360

What is claimed is:
 1. An isolated swine growth hormone secretagoguereceptor which comprises the amino acid sequence of SEQ ID NO:
 3. 2. Anisolated human growth hormone secretagogue receptor which comprises theamino acid sequence of SEQ ID NO:
 7. 3. An isolated rat growth hormonesecretagogue receptor which comprises the amino acid sequence of SEQ IDNO:
 16. 4. An isolated nucleic acid which encodes swine growth hormonesecretagogue receptor which is that of SEQ ID NO:
 1. 5. A vectorcomprising a nucleic acid which encodes a growth hormone secretagoguereceptor in accordance with claim
 4. 6. A vector according to claim 5which is selected from the group consisting of: plasmnids, modified,viruses, yeast artificial chromosomes, bacteriophages, cosmids andtransposable elements.
 7. A host cell comprising a vector according toclaim
 6. 8. An isolated nucleic acid which encodes human growth hormonesecretagogue receptor which is that of SEQ ID NOs: 6, 11 or
 13. 9. Avector comprising a nucleic acid which encodes a growth hormonesecretagogue receptor in accordance with claim
 8. 10. A vector accordingto claim 9 which is selected from the group consisting of: plasmids,modified viruses, yeast artificial chromosomes, bacteriophages, cosmidsand transposable elements.
 11. A host cell comprising a vector accordingto claim
 10. 12. An isolated nucleic acid which encodes rat growthhormone secretagogue receptor which is that of SEQ ID NO: 14 or SEQ IDNO:
 15. 13. A vector comprising a nucleic acid which encodes a growthhormone secretagogue receptor in accordance with claim
 12. 14. A vectoraccording to claim 13 which is selected from the group consisting of:plasmids, modified viruses, yeast artificial chromosomes,bacteriophages, cosmids and transposable elements.
 15. A host cellcomprising a vector according to claim
 14. 16. A growth hormonesecretagogue receptor, free from receptor-associated proteins whichcomprises the amino acid sequence of SEQ ID NO:
 3. 17. An isolated humangrowth hormone secretagogue receptor which comprises the amino acidsequence of SEQ ID NO:
 8. 18. An isolated human growth hormonesecretagogue receptor which comprises the amino acid sequence of SEQ IDNO:
 12. 19. An isolated human growth hormone secretagogue receptor whichcomprises the amino acid sequence of SEQ ID NO:
 13. 20. A growth hormonesecretagogue receptor, free from receptor-associated proteins whichcomprises the amino acid sequence of SEQ ID NO:
 7. 21. A growth hormonesecretagogue receptor, free from receptor-associated proteins whichcomprises the amino acid sequence of SEQ ID NO:
 8. 22. A growth hormonesecretagogue receptor, free from receptor-associated proteins whichcomprises the amino acid sequence of SEQ ID NO:
 12. 23. A growth hormonesecretagogue receptor, free from receptor-associated proteins whichcomprises the amino acid sequence of SEQ ID NO:
 13. 24. A growth hormonesecretagogue receptor, free from receptor-associated proteins whichcomprises the amino acid sequence of SEQ ID NO: 16.